Compositions and methods for reducing ice crystal formation

ABSTRACT

The present invention provides peptoid polymers capable of reducing or inhibiting the formation of ice crystals at sub 0° C. temperatures. Also provided are peptoid-peptide hybrids comprising the peptoid polymers provided herein. The peptoid polymers and peptoid-peptide hybrids provided herein are useful for making cryoprotectant solutions. The peptoid polymers, peptoid-peptide hybrids, and cryoprotectant solutions provided herein are useful for making antifreeze solutions, frozen food products, and cosmetic care products. Also provided herein are methods for preserving a tissue, an organ, a cell, or a biological macromolecule using the compositions described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/969,256 filed May 2, 2018, which is a continuation of U.S.application Ser. No. 15/486,522 filed Apr. 13, 2017, now U.S. Pat. No.9,986,733 issued Jun. 5, 2018, which is a continuation of InternationalApplication No. PCT/US2016/056852 filed Oct. 13, 2016, which claimspriority to U.S. Provisional Application No. 62/241,588 filed Oct. 14,2015, the disclosures of which are hereby incorporated by reference intheir entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Contract No.W81XWH16C0066 awarded by the Department of Defense, Defense HealthAgency. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Cryoprotective agents (CPAs) are compounds that when present in solutioncan reduce or inhibit ice crystal formation in solutions exposed to sub0° C. temperatures. Current CPAs include small molecules (often referredto as penetrating CPAs), synthetic polymers, and antifreeze proteins.

Organ transplantation is currently the best treatment for end-stageorgan failure in terms of survival, quality of life, and costeffectiveness. Unfortunately, a steep gap exists between supply anddemand of organ transplants, and is one of the major medical obstaclesthat forces patients of debilitating disease to suffer low quality oflife over a long period wait time. The apparent lack of organs is due toconsiderable waste from the absence of a reliable preservation method.In fact, over 50% of lungs, pancreas, and hearts remain unharvested fromdeceased donors.

In order to properly preserve organs, they have to be flushed with apreservation solution to remove blood and stabilize the organs. Evenonce stabilized in the preservation solution, there is only a limitedtime available for organ allocation, transportation, and transplantationafter removal from the donor (˜6-12 hours). This small timeframe resultsin most organs going to local patients because remote patient matchesoften cannot be confirmed in the limited time. As a result of thisshortage and in spite of laws which exist in almost all countriesprohibiting the sale of one's organs, illicit organ trade and humantrafficking has risen to supply demand.

Current penetrating CPAs used in organ preservation include ethyleneglycol, 1,2-propanediol, dimethyl sulfoxide, formamide, glycerol,sucrose, lactose, and D-mannitol, generally among others. In order toreduce or inhibit ice crystal growth at organ preservation temperatures,the effective concentration of the penetrating CPAs must be very high(≥60% is often required). At such high concentrations these compoundscan be toxic to the tissues they are attempting to preserve, and themassive removal of CPAs upon warming before transplantation can lead toirreversible cell death.

Other CPAs used to reduce or inhibit ice crystal formation includesynthetic polymers and antifreeze proteins. Similar to the penetratingCPAs, each of these have their drawbacks. Synthetic polymers, forexample, are not capable of permeating the cellular membrane. As such,synthetic polymer CPAs can only control extracellular ice formation. Inorder to effectively preserve the biological sample, ice crystalformation must be controlled both inside and outside the cell.Naturally-occurring antifreeze proteins, such as those isolated fromfish, plants, or insects, are highly effective at preventing iceformation, but current antifreeze proteins that are available are of lowpurity and are extremely expensive. Additionally, the use of antifreezeproteins to preserve a biological sample introduces a potential sourceof immunogenicity.

As such, there is a need in the art for novel non-toxic compounds toeffectively reduce or inhibit ice crystal formation at sub 0° C. andcryogenic temperatures. The present disclosure satisfies this need andprovides other advantages as well.

BRIEF SUMMARY OF THE INVENTION

In some aspects, provided herein is a peptoid polymer of formula (I):

a tautomer thereof or stereoisomer thereof,

wherein:

each R¹ is independently selected from the group consisting of H,optionally substituted C₁₋₁₈ alkyl, optionally substituted C₂₋₁₈alkenyl, optionally substituted C₂₋₁₈ alkynyl, optionally substitutedC₁₋₁₈ hydroxyalkyl, optionally substituted alkoxy, optionallysubstituted C₁₋₁₈ alkylamino, optionally substituted C₁₋₁₈ alkylthio,optionally substituted carboxyalkyl, C₃₋₁₀ cycloalkyl, heterocycloalkyl,aryl, heteroaryl, (C₃₋₁₀ cycloalkyl)alkyl, (heterocycloalkyl)alkyl,arylalkyl, and heteroarylalkyl;

wherein at least one instance of R¹ is C₁₋₁₈ hydroxyalkyl, and

wherein any of the cycloalkyl, heterocycloalkyl, aryl, and heteroarylgroups is optionally and independently substituted with one or more R³groups;

each R² is independently selected from the group consisting of H,optionally substituted C₁₋₁₈ alkyl, optionally substituted C₂₋₁₈alkenyl, optionally substituted C₂₋₁₈ alkynyl, optionally substitutedC₁₋₁₈ hydroxyalkyl, optionally substituted C₁₋₁₈ alkylamino, optionallysubstituted C₁₋₁₈ alkylthio, and optionally substituted carboxyalkyl;

each R³ is independently selected from the group consisting of halogen,oxo, thioxo, —OH, —SH, amino, C₁₋₈ alkyl, C₁₋₈ hydroxyalkyl, C₁₋₈alkylamino, and C₁₋₈ alkylthio;

X and Y are independently selected from the group consisting of H,optionally substituted C₁₋₈ alkylamino, —OH, —SH, carboxy, optionallysubstituted C₁₋₈ hydroxyalkyl, optionally substituted C₁₋₈ alkylamino,optionally substituted C₂₋₈ alkylthio, optionally substituted C₁₋₈carboxyalkyl, and halogen; or

alternatively X and Y are taken together to form a covalent bond; and

the subscript n, representing the number of monomers in the polymer, isbetween 2 and 50;

provided that all instances of R¹ are not ethylhydroxy when n is between3 and 7.

In some embodiments, each instance of R¹ in the peptoid polymer isselected from the group consisting of:

wherein:

m is between 1 and 8; and

R³ is selected from the group consisting of H, C₁₋₈ alkyl, hydroxyl,thiol, nitro, amine, oxo, and thioxo.

In some embodiments, each instance of R¹ in the peptoid polymer isselected from the group consisting of:

In some embodiments, each instance of R¹ in the peptoid polymer is aC₁₋₁₈ hydroxyalkyl group. In some embodiments, each instance of R¹ is aC₁₋₆ hydroxyalkyl group. In some embodiments, each instance of R¹ is thesame C₁₋₆ hydroxyalkyl group. In some embodiments, each instance of R¹is:

In some embodiments, each instance of R² is H.

In some embodiments, the sequence length of the peptoid polymer, n, isbetween 3 and 25. In some embodiments, the sequence length of thepeptoid polymer, n, is between 5 and 25. In some embodiments, thesequence length of the peptoid polymer, n, is between 8 and 50. In someembodiments, the sequence length of the peptoid polymer, n, is between 8and 20.

In some embodiments, X and Y are H, optionally substituted C₁₋₈alkylamino, —OH, —SH, carboxy, optionally substituted C₁₋₈ hydroxyalkyl,optionally substituted C₁₋₈ alkylamino, optionally substituted C₂₋₈alkylthio, optionally substituted C₁₋₈ carboxyalkyl, or halogen.

In some embodiments, X and Y of the peptoid polymer are taken togetherto form a covalent bond.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 3 Nhp monomers and 7 Nsbmonomers, 4 Nhp monomers and 6 Nsb monomers, 5 Nhp monomers and 5 Nsbmonomers, 6 Nhp monomers and 4 Nsb monomers, 7 Nhp monomers and 3 Nsbmonomers, 8 Nhp monomers and 2 Nsb monomers, or 10 Nhp monomers.

In some embodiments, the peptoid polymer has the sequenceNhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp (SEQ ID NO:2), and X is H orC₁₋₈ acyl and Y is —OH or —NH₂ or C₁₋₈ alkyl. In some embodiments, thepeptoid polymer has the sequence Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb(SEQ ID NO:1), and X is H or C₁₋₈ acyl and Y is —OH or —NH₂ or C₁₋₈alkyl. In some embodiments, the peptoid polymer has the sequenceNsb-Nhp-Nhp-Nhp-Nsb-Nhp-Nhp-Nhp-Nsb-Nhp (SEQ ID NO:7), and X is H orC₁₋₈ acyl and Y is —OH or —NH₂ or C₁₋₈ alkyl. In some embodiments, thepeptoid polymer has the sequence Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb(SEQ ID NO:8), and X is H or C₁₋₈ acyl and Y is —OH or —NH₂ or C₁₋₈alkyl. In some embodiments, the peptoid polymer has the sequenceNsb-Nhp-Nhp-Nhp-Nhp-Nhp-Nsb-Nhp-Nhp-Nhp (SEQ ID NO:9), and X is H orC₁₋₈ acyl and Y is —OH or —NH₂ or C₁₋₈ alkyl.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 3 Nhp monomers and 7 Nmemonomers, 4 Nhp monomers and 6 Nme monomers, 5 Nhp monomers and 5 Nmemonomers, 6 Nhp monomers and 4 Nme monomers, 7 Nhp monomers and 3 Nmemonomers, or 8 Nhp monomers and 2 Nme monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 5 Nhe monomers and 5 Nsbmonomers, or 5 Nhp monomers and 5 Nbu monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 4 Nhp monomers and 6 Nibmonomers, 4 Nhp monomers and 6 Nbu monomers, 4 Nhp monomers and 6 Nprmonomers, or 4 Nhp monomers and 6 Nip monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is14, and the peptoid polymer comprises: 6 Nhp monomers and 8 Nsbmonomers, 7 Nhp monomers and 7 Nsb monomers, 8 Nhp monomers and 6 Nsbmonomers, 10 Nhp monomers and 4 Nsb monomers, or 14 Nhp monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is14, and the peptoid polymer comprises: 6 Nhp monomers and 8 Nibmonomers, 7 Nhp monomers and 7 Nib monomers, 8 Nhp monomers and 6 Nibmonomers, 10 Nhp monomers and 4 Nib monomers, or 14 Nhp monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is16, and the peptoid polymer comprises: 5 Nhp monomers and 11 Nsbmonomers, 7 Nhp monomers and 9 Nsb monomers, 8 Nhp monomers and 8 Nsbmonomers, 10 Nhp monomers and 6 Nsb monomers, 12 Nhp monomers and 4 Nsbmonomers, or 16 Nhp monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is22, and the peptoid polymer comprises: 7 Nhp monomers and 15 Nsbmonomers, 10 Nhp monomers and 12 Nsb monomers, 11 Nhp monomers and 11Nsb monomers, 14 Nhp monomers and 8 Nsb monomers, 17 Nhp monomers and 5Nsb monomers, or 22 Nhp monomers.

In some embodiments, the polymer is selected from the group of polymersset forth in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table8, or Table 9.

In some embodiments, the peptoid polymer described herein forms ahelical structure.

In some embodiments, the peptoid polymer reduces or inhibits ice crystalformation at a temperature within about 0° C. to about −20° C. In otherembodiments, the peptoid polymer reduces or inhibits ice crystalformation at a temperature within about −20° C. to about −40° C. In someembodiments, the peptoid polymer reduces or inhibits ice crystalformation at about −20° C. In other embodiments, the peptoid polymerreduces or inhibits ice crystal formation at a temperature within about−40° C. to about −200° C. (e.g., −196° C.). In certain embodiments, theconcentration of the peptoid polymer (e.g., present in a composition,formulation, or product such as a cryoprotectant solution, antifreezesolution, frozen food product, or cosmetic care product) is betweenabout 100 nM and about 100 mM. In particular embodiments, theconcentration of the peptoid polymer is between about 1 and 10 mM (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM).

In another aspect, the present invention provides a peptoid-peptidehybrid comprising a peptoid polymer described herein and one or moreamino acids, wherein the one or more amino acids are located at one orboth ends of the peptoid polymer and/or between one or more peptoidmonomers. In some embodiments, the one or more amino acids are selectedfrom the group consisting of alanine, cysteine, aspartic acid, glutamicacid, phenylalanine, glycine, histidine, isoleucine, arginine, lysine,leucine, methionine, asparagine, proline, glutamine, serine, threonine,valine, tryptophan, tyrosine, and a combination thereof. In particularembodiments, the one or more amino acids are selected from the groupconsisting of isoleucine, leucine, serine, threonine, alanine, valine,arginine, and a combination thereof.

In another aspect, the present invention provides a cryoprotectantsolution comprising a peptoid polymer described herein, apeptoid-peptide hybrid described herein, or a combination thereof. Insome embodiments, the cryoprotectant solution further comprises acompound selected from the group consisting of an ionic species, apenetrating cryoprotectant, a non-penetrating cryoprotectant, anantioxidant, a cell membrane stabilizing compound, an aquaporin or otherchannel forming compound, an alcohol, a sugar, a sugar derivative, anonionic surfactant, a protein, dimethyl sulfoxide (DMSO), polyethyleneglycol (PEG), Ficoll®, polyvinylpyrrolidone, polyvinyl alcohol,hyaluronan, formamide, a natural or synthetic hydrogel, and acombination thereof.

In some instances, the cryoprotectant solution further comprises analcohol selected from the group consisting of propylene glycol, ethyleneglycol, glycerol, methanol, butylene glycol, adonitol, ethanol,trimethylene glycol, diethylene glycol, polyethylene oxide, erythritol,sorbitol, xythyritol, polypropylene glycol, 2-methyl-2,4-pentanediol(MPD), mannitol, inositol, dithioritol, 1,2-propanediol, and acombination thereof.

In some instances, the cryoprotectant solution further comprises a sugarthat is selected from the group consisting of a monosaccharide, adisaccharide, a polysaccharide, and a combination thereof. In someinstances, the sugar is a monosaccharide selected from the groupconsisting of glucose, galactose, arabinose, fructose, xylose, mannose,3-O-Methyl-D-glucopyranose, and a combination thereof. In otherinstances, the sugar is a disaccharide selected from the groupconsisting of sucrose, trehalose, lactose, maltose, and a combinationthereof. In still other instances, the sugar is a polysaccharideselected from the group consisting of raffinose, dextran, and acombination thereof.

In other instances, the cryoprotectant solution further comprises a PEGthat has an average molecular weight less than about 1,000 g/mol. Inparticular instances, the PEG has an average molecular weight betweenabout 200 and 400 g/mol.

In some instances, the cryoprotectant solution further comprises aprotein selected from the group consisting of bovine serum albumin,human serum albumin, gelatin, and a combination thereof. In otherinstances, the cryoprotectant solution further comprises a natural orsynthetic hydrogel that comprises chitosan, hyaluronic acid, or acombination thereof. In yet other instances, the cryoprotectant solutionfurther comprises a nonionic surfactant selected from the groupconsisting of polyoxyethylene lauryl ether, polysorbate 80, and acombination thereof.

In another aspect, provided herein is a method for preserving a tissue,organ, or cell. The method comprises contacting the tissue, organ, orcell with a peptoid polymer described herein, a peptoid-peptide hybriddescribed herein, a cryoprotectant solution described herein, or acombination thereof. In some embodiments, the tissue is a bioengineeredtissue. In some embodiments, the tissue, organ, or cell is selected fromthe group consisting of heart, liver, lung, kidney, pancreas, intestine,thymus, cornea, nerve cells, blood platelets, sperm cells, oocytes,embryonic cells, stem cells (e.g., human pluripotent stem cells,hematopoietic stem cells), lymphocytes, granulocytes, immune systemcells, bone cells, organoids, and a combination thereof.

In some embodiments, the peptoid polymer, peptoid-peptide hybrid,cryoprotectant solution, or combination thereof is present in an amountsufficient to reduce or inhibit ice crystal formation at a temperaturewithin about 0° C. to about −20° C. In other embodiments, the peptoidpolymer, peptoid-peptide hybrid, cryoprotectant solution, or combinationthereof is present in an amount sufficient to reduce or inhibit icecrystal formation at a temperature within about −20° C. to about −40° C.In some embodiments, the peptoid polymer, peptoid-peptide hybrid,cryoprotectant solution, or combination thereof is present in an amountsufficient to reduce or inhibit ice crystal formation at about −20° C.In other embodiments, the peptoid polymer, peptoid-peptide hybrid,cryoprotectant solution, or combination thereof is present in an amountsufficient to reduce or inhibit ice crystal formation at a temperaturewithin about −40° C. to about −200° C. (e.g., −196° C.). In certainembodiments, the concentration of the peptoid polymer and/orpeptoid-peptide hybrid in the cryoprotectant solution is between about100 nM and about 100 mM. In particular embodiments, the concentration ofthe peptoid polymer and/or peptoid-peptide hybrid in the cryoprotectantsolution is between about 1 and 10 mM (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 mM).

In yet another aspect, provided herein is a method for preserving abiological macromolecule. The method comprises contacting the biologicalmacromolecule with a peptoid polymer described herein, a peptoid-peptidehybrid described herein, a cryoprotectant solution described herein, ora combination thereof. In some embodiments, the biological macromoleculeis selected from the group consisting of a nucleic acid, an amino acid,a protein, an isolated protein, a peptide, a lipid, a compositestructure, and a combination thereof.

In another aspect, the present invention provides a cosmetic careproduct comprising a peptoid polymer described herein, a peptoid-peptidehybrid described herein, a cryoprotectant solution described herein, ora combination thereof.

In another aspect, the present invention provides an antifreeze productsuch as a deicing or ice-inhibiting product comprising a peptoid polymerdescribed herein, a peptoid-peptide hybrid described herein, acryoprotectant solution described herein, or a combination thereof. Insome embodiments, the antifreeze product is used to prevent, inhibit, ordelay the formation of ice on objects including, but not limited to,aircrafts or parts thereof, gas pipelines, windows, electricalequipment, drones, cables (e.g., power lines), mechanical equipment(e.g., car engines, gear systems, brake systems, etc.), and the like.

In still another aspect, the present invention provides a frozen foodproduct comprising a peptoid polymer described herein, a peptoid-peptidehybrid described herein, a cryoprotectant solution described herein, ora combination thereof. In some embodiments, the frozen food product isselected from the group consisting of ice cream, yogurt, seafood, fruit,and meat products.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a general protocol for the synthesis of peptoidoligomers using the “submonomer” approach.

FIGS. 2A and 2B show the results of a capillary tube freeze assay thatwas performed at −20° C. FIG. 2A illustrates the assay in whichCompounds 1 (1 eq.) and 10 (1 eq.) were dissolved in MilliQ water andsubjected to subzero temperatures. Comparison was made to water aloneand a solution of ethylene glycol (EG) (18 eq.). FIG. 2B displaysnormalized results of the assay depicted in FIG. 2A.

FIGS. 3A-3D show x-ray diffraction (XRD) crystallography data. FIG. 3Ashows XRD data for a solution containing 5 mM Compound 12 and 17.5%(v/v) ethylene glycol (EG). FIG. 3B shows XRD data for a solutioncontaining 30% (v/v) EG. FIG. 3C shows XRD data for a solutioncontaining 17.5% (v/v) EG. FIG. 3D shows ice ring scores for a number ofsolutions containing EG, Compound 2 (labeled as “B”; SEQ ID NO:10),Compound 12 (labeled as “D”; SEQ ID NO:8), and/or Compound 8 (labeled as“E”; SEQ ID NO:9). For each different solution, two separate ice ringscores were determined.

FIGS. 4A-4G show x-ray diffraction (XRD) crystallography data forsolutions containing 5 mg/mL of Compound 10, Compound 12, Compound 8,Compound 13, Compound 11, and Compound 58, compared to a ethylene glycol(EG) control. Each solution also contained 300 mM NaCl, 100 mM HEPES,15% (v/v) ethylene glycol, and pH was adjusted to 7.2. FIG. 4A: Compound10 XRD crystallography pattern (left) and spectrum plot (right). FIG.4B: Compound 12 XRD crystallography pattern (left) and spectrum plot(right). FIG. 4C: Compound 8 XRD crystallography pattern (left) andspectrum plot (right). FIG. 4D: Compound 13 XRD crystallography pattern(left) and spectrum plot (right). FIG. 4E: Compound 11 XRDcrystallography pattern (left) and spectrum plot (right). FIG. 4F:Compound 58 XRD crystallography pattern (left) and spectrum plot(right). FIG. 4G: EG control XRD crystallography pattern (left) andspectrum plot (right). For XRD spectrum plots, intensity was plotted asa function of angle (20 degrees).

FIGS. 5A-5G show x-ray diffraction (XRD) crystallography data forsolutions containing 1 mg/mL of Compound 10, Compound 12, Compound 8,Compound 13, Compound 11, and Compound 58, compared to a ethylene glycol(EG) control. Each solution also contained 300 mM NaCl, 100 mM HEPES,17.5% (v/v) ethylene glycol, and pH was adjusted to 7.2. FIG. 5A:Compound 10 XRD crystallography pattern (left) and spectrum plot(right). FIG. 5B: Compound 12 XRD crystallography pattern (left) andspectrum plot (right). FIG. 5C: Compound 8 XRD crystallography pattern(left) and spectrum plot (right). FIG. 5D: Compound 13 XRDcrystallography pattern (left) and spectrum plot (right). FIG. 5E:Compound 11 XRD crystallography pattern (left) and spectrum plot(right). FIG. 5F: Compound 58 XRD crystallography pattern (left) andspectrum plot (right). FIG. 5G: EG control XRD crystallography pattern(left) and spectrum plot (right). For XRD spectrum plots, intensity wasplotted as a function of angle (20 degrees).

FIGS. 6A-6C show two solutions that were flash frozen, rewarmed, andsubsequently refrozen. The control solution contained 22.5% (v/v)ethylene glycol (EG), while the test solution contained 22.5% EG and 5mg/mL (0.5% (w/v)) Compound 12. FIG. 6A shows that during rapid freezingin liquid nitrogen, the solution containing Compound 12 vitrified whilethe control solution completely froze. FIG. 6B shows that duringrewarming at 37° C., the solution containing Compound 12 unfroze (withintwo seconds) while the control stayed frozen. FIG. 6C shows that afterovernight in a −20° C. freezer, the Compound 12 solution remainedunfrozen, unlike the control.

FIG. 7 shows the results of a cell toxicity study performed on HEK 293cells in which Compound 12 (squares) or DMSO (circles) was added to cellculture media. A sample in which no Compound 12 or DMSO was added(“Culture Media” (triangles)) served as a control. Serial dilutions wereperformed in order to test different concentrations of Compound 12 andDMSO.

FIG. 8 shows the results of a cryopreservation assay performed on HEK293 cells, comparing a solution containing ethylene glycol (EG) to asolution containing EG and Compound 12. Cell viability was measured 12hours post-thaw.

FIG. 9 shows the results of a cryopreservation assay performed on HEK293 cells, comparing a solution containing 5 mg/mL of Compound 12 plus amixture of glycols, disaccharides, and a general buffer to solutionscontaining VS2E or M22. Cell viability was measured 16 hours post-thaw.Cell were vitrified with liquid nitrogen (LN2).

DETAILED DESCRIPTION OF THE INVENTION I. INTRODUCTION

The banking of cells and tissues at low temperatures usingcryopreservation is critical for many biological products andapplications, but remains a significant problem that has yet to allowthe successful full recovery or viable therapeutic cells, tissues, andorgans. Cryopreservation is typically performed with cryoprotectiveagents (CPAs), which are critical chemical additives such as dimethylsulfoxide (DMSO), bovine serum albumin (BSA), and others. The CPAs areused to improve the post-thaw viability of cryopreserved biologicalsystems by preventing ice crystal nucleation and growth. However, theseagents exhibit various levels of cytotoxicity at their effectiveconcentrations and thus limit the success of cryopreservation,biobanking, and advanced regenerative medicine. This lack of aneffective and safe CPA contributes to the widespread use of toxic CPAs.Beyond biological products and applications, preventing ice formationremains a physical and chemical problem for a wide variety of industriesand technology sectors.

The present invention is based, in part, on the surprising discoverythat N-substituted biomimetic amino acid polymers (peptoids) andpeptoid-peptide hybrids have ice crystallization inhibition properties.Provided herein are polymers for reducing or inhibiting ice crystalformation at sub 0° C. and cryogenic temperatures. These polymers areuseful in making cryoprotectant solutions. Also provided herein aremethods for preserving a tissue, organ, or cell using cryoprotectantsolutions comprising the peptoid polymers described herein.Additionally, cosmetic care, deicing, and frozen food products withantifreeze properties comprising the peptoid polymers described hereinare provided. Upon reading the detailed description, a person ofordinary skill in the art will recognize there are other advantages thatflow from the teachings provided herein.

II. ABBREVIATIONS AND DEFINITIONS

The abbreviations used herein are conventional, unless otherwisedefined. The following abbreviations are used to refer to the monomerunits of the peptoid polymer: Nsb (2-(sec-butylamino)acetic acid), Nib(2-(isobutylamino)acetic acid), Nbu (2-butylamino)acetic acid), Npr(2-propylamino)acetic acid), Nip (2-(isopropylamino)acetic acid), Nme(2-(methylamino)acetic acid), Nhp (2-((2-hydroxypropyl)amino)aceticacid), Nhe (2-((2-hydroxyethyl)amino)acetic acid), Ndp(2-((2,3-dihydroxypropryl)amino)acetic acid, Nyp(2-((1-hydroxypropan-2-yl)amino) acetic acid), Nep(2-((1-(4-hydroxyphenyl)ethyl)amino) acetic acid, Ndh(2-((1,3,-dihydrooxypropan-2-yl)amino)acetic acid, and Nop(2-((3-(2-oxopyrrolindin-l-yl)propyl)amino)acetic acid. The followingabbreviations are used to refer to chemical compounds: DMF (N,N′-dimethylformamide), DIEA (diisopropylethylamine, DIC(N,N′-diisopropylcarbodiimide), ACN (acetonitrile), DCM (methylenechloride), HFIP (hexafluoroisopropyl alcohol); Fmoc(9-fluorenylmethoxycarbonyl).

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forinstance, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the agent” includes reference to one or more agents knownto those skilled in the art, and so forth.

The term “about” as used herein to modify a numerical value indicates adefined range around that value. If “X” were the value, “about X” wouldindicate a value from 0.9X to 1.1X, and more preferably, a value from0.95X to 1.05X. Any reference to “about X” specifically indicates atleast the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X,1.03X, 1.04X, and 1.05X. Thus, “about X” is intended to teach andprovide written description support for a claim limitation of, e.g.,“0.98X.”

“Alkyl” refers to a straight or branched, saturated, aliphatic radicalhaving the number of carbon atoms indicated. Alkyl can include anynumber of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈,C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ andC₅₋₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groupshaving up to 30 carbons atoms, such as, but not limited to heptyl,octyl, nonyl, decyl, etc. Alkyl groups can be substituted orunsubstituted. Alkyl groups can be optionally substituted with one ormore moieties selected from halo, hydroxy, amino, thiol, alkylamino,alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

“Alkenyl” refers to a straight chain or branched hydrocarbon having atleast 2 carbon atoms and at least one double bond. Alkenyl can includeany number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈,C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆.Alkenyl groups can have any suitable number of double bonds, including,but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groupsinclude, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl,1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl,isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl,2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenyl groups can be substitutedor unsubstituted. Alkenyl groups can be optionally substituted with oneor more moieties selected from halo, hydroxy, amino, thiol, alkylamino,alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

“Alkynyl” refers to either a straight chain or branched hydrocarbonhaving at least 2 carbon atoms and at least one triple bond. Alkynyl caninclude any number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇,C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₅, C₄₋₆, C₅, C₅₋₆, andC₆. Examples of alkynyl groups include, but are not limited to,acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl,butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl,1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl,1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl.Alkynyl groups can be substituted or unsubstituted. Alkynyl groups canbe optionally substituted with one or more moieties selected from halo,hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido,nitro, oxo, thioxo, and cyano.

“Alkylene” refers to a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated, and linking atleast two other groups, i.e., a divalent hydrocarbon radical. The twomoieties linked to the alkylene can be linked to the same atom ordifferent atoms of the alkylene group. For instance, a straight chainalkylene can be the bivalent radical of —(CH₂)_(n)—, where n is anynumber of suitable carbon atoms. Representative alkylene groups include,but are not limited to, methylene, ethylene, propylene, isopropylene,butylene, isobutylene, sec-butylene, pentylene and hexylene. Alkylenegroups can be substituted or unsubstituted. Alkylene groups can beoptionally substituted with one or more moieties selected from halo,hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido,nitro, oxo, thioxo, and cyano.

“Alkenylene” refers to an alkenyl group, as defined above, linking atleast two other groups, i.e., a divalent hydrocarbon radical. The twomoieties linked to the alkenylene can be linked to the same atom ordifferent atoms of the alkenylene. Alkenylene groups include, but arenot limited to, ethenylene, propenylene, isopropenylene, butenylene,isobutenylene, sec-butenylene, pentenylene and hexenylene. Alkenylengroups can be substituted or unsubstituted. Alkenylene groups can beoptionally substituted with one or more moieties selected from halo,hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido,nitro, oxo, thioxo, and cyano.

“Alkynylene” refers to an alkynyl group, as defined above, linking atleast two other groups, i.e., a divalent hydrocarbon radical. The twomoieties linked to the alkynylene can be linked to the same atom ordifferent atoms of the alkynylene. Alkynylene groups include, but arenot limited to, ethynylene, propynylene, isopropynylene, butynylene,sec-butynylene, pentynylene and hexynylene. Alkynylene groups can besubstituted or unsubstituted. Alkynylene groups can be optionallysubstituted with one or more moieties selected from halo, hydroxy,amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo,thioxo, and cyano.

“Halogen” or “halo” refers to fluorine, chlorine, bromine and iodine.

“Amine” or “amino” refers to an —N(R)₂ group where the R groups can behydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,or heteroaryl, among others. The R groups can be the same or different.The amino groups can be primary (each R is hydrogen), secondary (one Ris hydrogen) or tertiary (each R is other than hydrogen). The alkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroarylgroups can be optionally substituted with one or more moieties selectedfrom halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl,carboxy, amido, nitro, oxo, thioxo, and cyano.

“Hydroxyl” or “hydroxy” refers to an —OH group. The hydroxyl can be atany suitable carbon atom.

“Thiol” refers to an —SH group. The thiol group can be at any suitablecarbon atom.

“Oxo” refers to a double bonded O group (═O, —C(O)—). The oxo group canbe at any suitable carbon atom.

“Thioxo” refers to a double bonded S group (═S). The thioxo group can beat any suitable carbon atom.

“Nitro” refers to a —NO₂ group. The nitro group can be at any suitablecarbon atom.

“Carboxy” refers to a carboxylic acid group of the formula —C(O)OH or—CO₂H.

“Cycloalkyl” refers to a saturated or partially unsaturated, monocyclic,fused bicyclic or bridged polycyclic ring assembly containing from 3 to12 ring atoms, or the number of atoms indicated. Cycloalkyl can includeany number of carbons, such as C₃₋₆, C₄₋₆, C₅₋₆, C₃₋₈, C₄₋₈, C₅₋₈, C₆₋₈,C₃₋₉, C₃₋₁₀, C₃₋₁₁, and C₃₋₁₂. Saturated monocyclic cycloalkyl ringsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl ringsinclude, for example, norbornane, [2.2.2] bicyclooctane,decahydronaphthalene and adamantane. Cycloalkyl groups can also bepartially unsaturated, having one or more double or triple bonds in thering. Representative cycloalkyl groups that are partially unsaturatedinclude, but are not limited to, cyclobutene, cyclopentene, cyclohexene,cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene,cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene,and norbornadiene. When cycloalkyl is a saturated monocyclic C₃₋₈cycloalkyl, exemplary groups include, but are not limited tocyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl andcyclooctyl. When cycloalkyl is a saturated monocyclic C₃₋₆ cycloalkyl,exemplary groups include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can besubstituted or unsubstituted. Cycloalkyl groups can be optionallysubstituted with one or more moieties selected from alkyl, alkenyl,alkynyl, halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy,amido, thiol, nitro, oxo, thioxo, and cyano. For example, cycloalkylgroups can be substituted with C₁₋₆ alkyl or oxo (═O), among manyothers.

“Heterocycloalkyl” refers to a saturated ring system having from 3 to 12ring members and from 1 to 4 heteroatoms of N, O and S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can also be oxidized, such as, but not limitedto, —S(O)— and —S(O)₂—. Heterocycloalkyl groups can include any numberof ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8,6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitablenumber of heteroatoms can be included in the heterocycloalkyl groups,such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3to 4. The heterocycloalkyl group can include groups such as aziridine,azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine,pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and 1,4-isomers),oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane,thiirane, thietane, thiolane (tetrahydrothiophene), thiane(tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine,isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine,dioxane, or dithiane. The heterocycloalkyl groups can also be fused toaromatic or non-aromatic ring systems to form members including, but notlimited to, indoline. Heterocycloalkyl groups can be unsubstituted orsubstituted. Heterocycloalkyl groups can be optionally substituted withone or more moieties selected from alkyl, alkenyl, alkynyl, halo,hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido,nitro, oxo, thioxo, and cyano. For example, heterocycloalkyl groups canbe substituted with C₁₋₆ alkyl or oxo (═O), among many others.

The heterocycloalkyl groups can be linked via any position on the ring.For example, aziridine can be 1- or 2-aziridine, azetidine can be 1- or2-azetidine, pyrrolidine can be 1-, 2- or 3-pyrrolidine, piperidine canbe 1-, 2-, 3- or 4-piperidine, pyrazolidine can be 1-, 2-, 3-, or4-pyrazolidine, imidazolidine can be 1-, 2-, 3- or 4-imidazolidine,piperazine can be 1-, 2-, 3- or 4-piperazine, tetrahydrofuran can be 1-or 2-tetrahydrofuran, oxazolidine can be 2-, 3-, 4- or 5-oxazolidine,isoxazolidine can be 2-, 3-, 4- or 5-isoxazolidine, thiazolidine can be2-, 3-, 4- or 5-thiazolidine, isothiazolidine can be 2-, 3-, 4- or5-isothiazolidine, and morpholine can be 2-, 3- or 4-morpholine.

When heterocycloalkyl includes 3 to 8 ring members and 1 to 3heteroatoms, representative members include, but are not limited to,pyrrolidine, piperidine, tetrahydrofuran, oxane, tetrahydrothiophene,thiane, pyrazolidine, imidazolidine, piperazine, oxazolidine,isoxazolidine, thiazolidine, isothiazolidine, morpholine,thiomorpholine, dioxane and dithiane. Heterocycloalkyl can also form aring having 5 to 6 ring members and 1 to 2 heteroatoms, withrepresentative members including, but not limited to, pyrrolidine,piperidine, tetrahydrofuran, tetrahydrothiophene, pyrazolidine,imidazolidine, piperazine, oxazolidine, isoxazolidine, thiazolidine,isothiazolidine, and morpholine.

“Aryl” refers to an aromatic ring system having any suitable number ofring atoms and any suitable number of rings. Aryl groups can include anysuitable number of ring atoms, such as, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6 to 14 ringmembers. Aryl groups can be monocyclic, fused to form bicyclic ortricyclic groups, or linked by a bond to form a biaryl group.Representative aryl groups include phenyl, naphthyl and biphenyl. Otheraryl groups include benzyl, having a methylene linking group. Some arylgroups have from 6 to 12 ring members, such as phenyl, naphthyl orbiphenyl. Other aryl groups have from 6 to 10 ring members, such asphenyl or naphthyl. Some other aryl groups have 6 ring members, such asphenyl. Aryl groups can be substituted or unsubstituted. Aryl groups canbe optionally substituted with one or more moieties selected from alkyl,alkenyl, alkynyl, halo, hydroxy, amino, thiol, alkylamino, alkoxy,haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

“Heteroaryl” refers to a monocyclic or fused bicyclic or tricyclicaromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5of the ring atoms are a heteroatom such as N, O or S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can also be oxidized, such as, but not limitedto, —S(O)— and —S(O)₂—. Heteroaryl groups can include any number of ringatoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8,3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitable numberof heteroatoms can be included in the heteroaryl groups, such as 1, 2,3, 4, or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3to 4, or 3 to 5. Heteroaryl groups can have from 5 to 8 ring members andfrom 1 to 4 heteroatoms, or from 5 to 8 ring members and from 1 to 3heteroatoms, or from 5 to 6 ring members and from 1 to 4 heteroatoms, orfrom 5 to 6 ring members and from 1 to 3 heteroatoms. The heteroarylgroup can include groups such as pyrrole, pyridine, imidazole, pyrazole,triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-,1,2,4- and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole,oxazole, and isoxazole. The heteroaryl groups can also be fused toaromatic ring systems, such as a phenyl ring, to form members including,but not limited to, benzopyrroles such as indole and isoindole,benzopyridines such as quinoline and isoquinoline, benzopyrazine(quinoxaline), benzopyrimidine (quinazoline), benzopyridazines such asphthalazine and cinnoline, benzothiophene, and benzofuran. Otherheteroaryl groups include heteroaryl rings linked by a bond, such asbipyridine. Heteroaryl groups can be substituted or unsubstituted.Heteroaryl groups can be optionally substituted with one or moremoieties selected from alkyl, alkenyl, alkynyl, halo, hydroxy, amino,thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo,thioxo, and cyano.

The heteroaryl groups can be linked via any position on the ring. Forexample, pyrrole includes 1-, 2- and 3-pyrrole, pyridine includes 2-, 3-and 4-pyridine, imidazole includes 1-, 2-, 4- and 5-imidazole, pyrazoleincludes 1-, 3-, 4- and 5-pyrazole, triazole includes 1-, 4- and5-triazole, tetrazole includes 1- and 5-tetrazole, pyrimidine includes2-, 4-, 5- and 6-pyrimidine, pyridazine includes 3- and 4-pyridazine,1,2,3-triazine includes 4- and 5-triazine, 1,2,4-triazine includes 3-,5- and 6-triazine, 1,3,5-triazine includes 2-triazine, thiopheneincludes 2- and 3-thiophene, furan includes 2- and 3-furan, thiazoleincludes 2-, 4- and 5-thiazole, isothiazole includes 3-, 4- and5-isothiazole, oxazole includes 2-, 4- and 5-oxazole, isoxazole includes3-, 4- and 5-isoxazole, indole includes 1-, 2- and 3-indole, isoindoleincludes 1- and 2-isoindole, quinoline includes 2-, 3- and 4-quinoline,isoquinoline includes 1-, 3- and 4-isoquinoline, quinazoline includes 2-and 4-quinoazoline, cinnoline includes 3- and 4-cinnoline,benzothiophene includes 2- and 3-benzothiophene, and benzofuran includes2- and 3-benzofuran.

Some heteroaryl groups include those having from 5 to 10 ring membersand from 1 to 3 ring atoms including N, O or S, such as pyrrole,pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine,pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene,furan, thiazole, isothiazole, oxazole, isoxazole, indole, isoindole,quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine,cinnoline, benzothiophene, and benzofuran. Other heteroaryl groupsinclude those having from 5 to 8 ring members and from 1 to 3heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole,pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, andisoxazole. Some other heteroaryl groups include those having from 9 to12 ring members and from 1 to 3 heteroatoms, such as indole, isoindole,quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine,cinnoline, benzothiophene, benzofuran and bipyridine. Still otherheteroaryl groups include those having from 5 to 6 ring members and from1 to 2 ring atoms including N, O or S, such as pyrrole, pyridine,imidazole, pyrazole, pyrazine, pyrimidine, pyridazine, thiophene, furan,thiazole, isothiazole, oxazole, and isoxazole.

“(Cycloalkyl)alkyl” refers to a radical having an alkyl component and acycloalkyl component, where the alkyl component links the cycloalkylcomponent to the point of attachment. The alkyl component is as definedabove, except that the alkyl component is at least divalent, analkylene, to link to the cycloalkyl component and to the point ofattachment. The alkyl component can include any number of carbons, suchas C₁₋₆, C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅,C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. The cycloalkyl component is as definedwithin. Exemplary (cycloalkyl)alkyl groups include, but are not limitedto, methyl-cyclopropyl, methyl-cyclobutyl, methyl-cyclopentyl andmethyl-cyclohexyl.

“(Heterocycloalkyl)alkyl” refers to a radical having an alkyl componentand a heterocycloalkyl component, where the alkyl component links theheterocycloalkyl component to the point of attachment. The alkylcomponent is as defined above, except that the alkyl component is atleast divalent, an alkylene, to link to the heterocycloalkyl componentand to the point of attachment. The alkyl component can include anynumber of carbons, such as C₀₋₆, C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₂₋₃,C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. Theheterocycloalkyl component is as defined above. (Heterocycloalkyl)alkylgroups can be substituted or unsubstituted.

“Arylalkyl” refers to a radical having an alkyl component and an arylcomponent, where the alkyl component links the aryl component to thepoint of attachment. The alkyl component is as defined above, exceptthat the alkyl component is at least divalent, an alkylene, to link tothe aryl component and to the point of attachment. The alkyl componentcan include any number of carbons, such as C₀₋₆, C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅,C₁₋₆, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. Thearyl component is as defined above. Examples of arylalkyl groupsinclude, but are not limited to, benzyl and ethyl-benzene. Arylalkylgroups can be substituted or unsubstituted.

“Heteroarylalkyl” refers to a radical having an alkyl component and aheteroaryl component, where the alkyl component links the heteroarylcomponent to the point of attachment. The alkyl component is as definedabove, except that the alkyl component is at least divalent, analkylene, to link to the heteroaryl component and to the point ofattachment. The alkyl component can include any number of carbons, suchas C₀₋₆, C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄,C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. The heteroaryl component is as definedwithin. Heteroarylalkyl groups can be substituted or unsubstituted.

“Carboxyalkyl” refers to a carboxy group linked to an alkyl, asdescribed above, and generally having the formula —C₁₋₈ alkyl-C(O)OH.Any suitable alkyl chain is useful. Carboxyalkyl groups can beoptionally substituted with one or more moieties selected from halo,hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido,nitro, oxo, thioxo, and cyano.

“Acyl” refers to an alkyl that contains an oxo substituted carbon at thepoint of attachment (—C(O)—C₁₋₈ alkyl). Any suitable alkyl chain isuseful. Acyl groups can be optionally substituted with one or moremoieties selected from halo, hydroxy, amino, thiol, alkylamino, alkoxy,haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

“Hydroxyalkyl” refers to an alkyl group, as defined above, where atleast one of the hydrogen atoms is replaced with a hydroxy group. As forthe alkyl group, hydroxyalkyl groups can have any suitable number ofcarbon atoms, such as C₁₋₆. Exemplary hydroxyalkyl groups include, butare not limited to, hydroxy-methyl, hydroxyethyl (where the hydroxy isin the 1- or 2-position), hydroxypropyl (where the hydroxy is in the 1-,2- or 3-position), hydroxybutyl (where the hydroxy is in the 1-, 2-, 3-or 4-position), hydroxypentyl (where the hydroxy is in the 1-, 2-, 3-,4- or 5-position), hydroxyhexyl (where the hydroxy is in the 1-, 2-, 3-,4-, 5- or 6-position), 1,2-dihydroxyethyl, and the like. Hydroxyalkylgroups can be optionally substituted with one or more moieties selectedfrom halo, thiol, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido,nitro, oxo, thioxo, and cyano. One of skill in the art will appreciatethat other hydroxyalkyl groups are useful in the present invention.

“Alkoxy” refers to an alkyl group having at least one bridging oxygenatom. The bridging oxygen atom can be anywhere within the alkyl chain(alkyl-O-alkyl) or the bridging oxygen atom can connect the alkyl groupto the point of attachment (alkyl-O—). In some instances, the alkoxycontains 1, 2, 3, 4, or 5 bridging oxygen atoms. As for alkyl group,alkoxy groups can have any suitable number of carbon atoms, such asC₁₋₂, C₁₋₄, and C₁₋₆. Alkoxy groups include, for example, methoxy,ethoxy, propoxy, iso-propoxy, methyloxy-ethyloxy-ethyl (C₁—O—C₂—O—C₂—),etc. One example of an alkoxy group is polyethylene glycol (PEG) whereinthe polyethylene glycol chain can include between 2 to 20 ethyleneglycol monomers. Alkoxy groups can be optionally substituted with one ormore moieties selected from halo, hydroxy, amino, thiol, alkylamino,haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano. Alkoxy groupscan be substituted or unsubstituted.

“Alkylamino” refers to an alkyl group as defined within, having one ormore amino groups. The amino groups can be primary, secondary ortertiary. Alkylamino groups useful in the present invention include, butare not limited to, ethyl amine, propyl amine, isopropyl amine, ethylenediamine and ethanolamine. The amino group can link the alkylamino to thepoint of attachment with the rest of the compound, be at any position ofthe alkyl group, or link together at least two carbon atoms of the alkylgroup. Alkylamino groups can be optionally substituted with one or moremoieties selected from halo, hydroxy, thiol, alkylamino, alkoxy,haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano. One of skillin the art will appreciate that other alkylaminos are useful in thepresent invention.

“Alkylthio” refers to an alkyl group as defined within, having one ormore thiol groups. Alkylthio groups useful in the present inventioninclude, but are not limited to, ethyl thiol, propyl thiol, andisopropyl thiol. The thiol group can link the alkylthio to the point ofattachment with the rest of the compound, be at any position of thealkyl group, or link together at least two carbon atoms of the alkylgroup. Alkylthio groups can be optionally substituted with one or moremoieties selected from halo, hydroxy, amino, alkylamino, alkoxy,haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano. One of skillin the art will appreciate that other alkylthio are useful in thepresent invention.

The term “wavy line” signifies the point of attachment of thesubstituent to the remainder of the molecule. When the wavy line is notdepicted as being specifically appended to a specific ring atom, thepoint of attachment can be to any suitable atom of the substituent. Forexample, the wavy line in the following structure:

is intended to include, as the point of attachment, any of thesubstitutable atoms.

The term “regenerative medicine” refers to a branch of medicine thatdeals with the process of replacing, engineering or regenerating humancells, tissues, or organs to restore or establish normal function. Insome embodiments, regenerative medicine includes growing tissues andorgans in the laboratory and safely implanting them when the body cannotheal itself

The term “bioengineered tissue” refers to one or more syntheticallycreated cells, tissues, or organs created for the purposes ofregenerative medicine. In some embodiments, bioengineered tissue refersto cells, tissues, or organs that were developed in the laboratory. Insome embodiments, bioengineered tissues refers to laboratory derivedheart, liver, lung, kidney, pancreas, intestine, thymus, cornea, stemcells (e.g., human pluripotent stem cells, hematopoietic stem cells),lymphocytes, granulocytes, immune system cells, bone cells, organoids,embryonic cells, oocytes, sperm cells, blood platelets, nerve cells, ora combination thereof.

The term “cryoprotectant solution” refers to a solution used to reduceor prevent freezing damage caused by ice crystal formation. In someembodiments, the cryoprotectant solution comprises one or more peptoidpolymers described herein. In other embodiments, the cryoprotectantsolution comprises one or more peptoid polymers and one or morepeptoid-peptide hybrids described herein. In some embodiments, thecryoprotectant solution protects a biological sample from freezingdamage. In some embodiments, the cryoprotectant solution protects anon-biological sample from ice crystal formation. In some embodiments,the cryoprotectant solution preserves a biological sample for an amountof time longer than if the biological sample were not exposed to reducedtemperatures.

The terms “vitrify” and “vitrification” mean the transformation of asubstance into a glass (i.e., non-crystalline amorphous solid). In thecontext of water, vitrification refers to the transformation of waterinto a glass without the formation of ice crystals, as opposed toordinary freezing, which results in ice crystal formation. Vitrificationis often achieved through very rapid cooling and/or the introduction ofagents that suppress ice crystal formation. On the other hand,“devitrify” and “devitrification” refer to the process ofcrystallization in a previously crystal-free (amorphous) glass. In thecontext of water ice, devitrification can mean the formation of icecrystals as the previously non-crystalline amorphous solid undergoesmelting.

The term “peptoid” refers to a polyamide of between about 2 and 1,000(e.g., between about 2 and 1,000, 2 and 950, 2 and 900, 2 and 850, 2 and800, 2 and 750, 2 and 700, 2 and 650, 2 and 600, 2 and 550, 2 and 500, 2and 450, 2 and 400, 2 and 350, 2 and 300, 2 and 250, 2 and 200, 2 and150, 2 and 100, 2 and 90, 2 and 80, 2 and 70, 2 and 60, 2 and 50, 2 and40, 2 and 30, 2 and 20, 2 and 10, 2 and 9, 2 and 8, 2 and 7, 2 and 6, 2and 5, 2 and 4, or 2 and 3) units having substituents “R¹” on the amidenitrogen atoms. Optionally, a second, independently selected,substituent “R²” can be attached to the carbon atom that is α- to thecarbonyl group (i.e., attached to the α-carbon atom). R² can be, but isnot limited to, H. In particular instances, a peptoid is a syntheticanalog of a peptide wherein the side chains that would otherwise beattached to the α-carbon atoms are instead attached to the amidenitrogen atoms. Peptoids are synthetic polymers with controlledsequences and lengths that can be made by automated solid-phase organicsynthesis to include a wide variety of side-chains having differentchemical functions. R¹-groups bonded to the amide nitrogen atoms in thepeptoids can include, but are not limited to, H, optionally substitutedC₁₋₁₈ alkyl, optionally substituted C₂₋₁₈ alkenyl, optionallysubstituted C₂₋₁₈ alkynyl, optionally substituted C₁₋₁₈ hydroxyalkyl,optionally substituted alkoxy, optionally substituted C₁₋₁₈ alkylamino,optionally substituted C₁₋₁₈ alkylthio, optionally substitutedcarboxyalkyl, C₃₋₁₀ cycloalkyl, heterocycloalkyl, aryl, heteroaryl,(C₃₋₁₀ cycloalkyl)alkyl, (heterocycloalkyl)alkyl, arylalkyl, andheteroarylalkyl groups, wherein any of the cycloalkyl, heterocycloalkyl,aryl, or heteroaryl groups is optionally and independently substitutedwith one or more “R³” groups. Each R³ group can be independentlyselected from halogen, oxo, thioxo, —OH, —SH, amino, C₁₋₈ alkyl, C₁₋₈hydroxyalkyl, C₁₋₈ alkylamino, or C₁₋₈ alkylthio groups. Furthermore,R¹-groups can comprise the side chain of any of the amino acids alanine(Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu),phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile),arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met),asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser),threonine (Thr), valine (Val), tryptophan (Trp), or tyrosine (Tyr).

The term “peptoid-peptide hybrid” refers to an oligomer that is composedof both peptoid monomer units and alpha amino acids (i.e., peptideunits).

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues, oran assembly of multiple polymers of amino acid residues.

The term “amino acid” includes but is not limited to naturally-occurringα-amino acids and their stereoisomers. “Stereoisomers” of amino acidsrefers to mirror image isomers of the amino acids, such as L-amino acidsor D-amino acids. For example, a stereoisomer of a naturally-occurringamino acid refers to the mirror image isomer of the naturally-occurringamino acid (i.e., the D-amino acid).

Naturally-occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified (e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine).Naturally-occurring a-amino acids include, without limitation, alanine(Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu),phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile),arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met),asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser),threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), andcombinations thereof. Stereoisomers of a naturally-occurring α-aminoacids include, without limitation, D-alanine (D-Ala), D-cysteine(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu),D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile),D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine(D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln),D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan(D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. For example, an L-aminoacid may be represented herein by its commonly known three letter symbol(e.g., Arg for L-arginine) or by an upper-case one-letter amino acidsymbol (e.g., R for L-arginine). A D-amino acid may be representedherein by its commonly known three letter symbol (e.g., D-Arg forD-arginine) or by a lower-case one-letter amino acid symbol (e.g., r forD-arginine).

III. DETAILED DECRIPTION OF THE EMBODIMENTS

Provided herein are peptoid polymers and methods for reducing orinhibiting ice crystal formation at sub 0° C. temperatures and cryogenictemperatures.

A. Peptoid Polymers

In some aspects, provided herein is a peptoid polymer of formula (I):

a tautomer thereof or stereoisomer thereof,

wherein:

each R¹ is independently selected from the group consisting of H,optionally substituted C₁₋₁₈ alkyl, optionally substituted C₂₋₁₈alkenyl, optionally substituted C₂₋₁₈ alkynyl, optionally substitutedC₁₋₁₈ hydroxyalkyl, optionally substituted alkoxy, optionallysubstituted C₁₋₁₈ alkylamino, optionally substituted C₁₋₁₈ alkylthio,optionally substituted carboxyalkyl, C₃₋₁₀ cycloalkyl, heterocycloalkyl,aryl, heteroaryl, (C₃₋₁₀ cycloalkyl)alkyl, (heterocycloalkyl)alkyl,arylalkyl, and heteroarylalkyl,

wherein at least one instance of R¹ is C₁₋₁₈ hydroxyalkyl, and

wherein any of the cycloalkyl, heterocycloalkyl, aryl, and heteroarylgroups is optionally and independently substituted with one or more R³groups;

each R² is independently selected from the group consisting of H,optionally substituted C₁₋₁₈ alkyl, optionally substituted C₂₋₁₈alkenyl, optionally substituted C₂₋₁₈ alkynyl, optionally substitutedC₁₋₁₈ hydroxyalkyl, optionally substituted C₁₋₁₈ alkylamino, optionallysubstituted C₁₋₁₈ alkylthio, and optionally substituted carboxyalkyl;

each R³ is independently selected from the group consisting of halogen,oxo, thioxo, —OH, —SH, amino, C₁₋₈ alkyl, C₁₋₈ hydroxyalkyl, C₁₋₈alkylamino, and C₁₋₈ alkylthio;

X and Y are independently selected from the group consisting of H,optionally substituted C₁₋₈ alkyl, optionally substituted C₁₋₈ acyl,optionally substituted C₁₋₈ alkylamino, —OH, —SH, —NH₂, carboxy,optionally substituted C₁₋₈ hydroxyalkyl, optionally substituted C₁₋₈alkylamino, optionally substituted C₂₋₈ alkylthio, optionallysubstituted C₁₋₈ carboxyalkyl, and halogen, or

alternatively X and Y are taken together to form a covalent bond; and

the subscript n, representing the number of monomers in the polymer, isbetween 2 and 50;

provided that all instances of R¹ are not hydroxyethyl when n is between3 and 7.

In some embodiments, each instance of R¹ in the peptoid polymer isselected from the group consisting of:

wherein: m is between 1 and 8; and R³ is selected from the groupconsisting of H, C₁₋₈ alkyl, hydroxyl, thiol, nitro, amine, oxo, andthioxo. In some embodiments, the repeating unit, m, can be between 1 and2, 1 and 3, 1 and 4, 1 and 5, 1 and 6, or 1 and 7. In some embodiments,the repeating unit, m, is 1, 2, 3, 4, 5, 6, 7, or 8.

In some embodiments, one or more R¹ monomers has a structure accordingto R^(1a):

In some embodiments, each R^(1a) group is independently selected from

In some embodiments, a mixture of the two stereoisomers are chosen. Insome embodiments, only the R stereoisomer of the monomer is chosen. Insome embodiments, only the S stereoisomer of this monomer is chosen.

In some embodiments, one or more R¹ monomers has a structure accordingto R^(1b):

In some embodiments, each R^(1b) group is independently selected from

In some embodiments, a mixture of the two stereoisomers are chosen. Insome embodiments, only the R stereoisomer of the monomer is chosen. Insome embodiments, only the S stereoisomer of this monomer is chosen.

In some embodiments, one or more R¹ monomers has a structure accordingto R^(1c):

In some embodiments, each R^(1c) group is independently selected from

In some embodiments, a mixture of the two stereoisomers are chosen. Insome embodiments, only the R stereoisomer of the monomer is chosen. Insome embodiments, only the S stereoisomer of this monomer is chosen.

In some embodiments, one or more R¹ monomers has a structure accordingto R^(1d):

In some embodiments, each R^(1d) group is independently selected from

In some embodiments, a mixture of the two stereoisomers are chosen. Insome embodiments, only the R stereoisomer of the monomer is chosen. Insome embodiments, only the S stereoisomer of this monomer is chosen.

In some embodiments, one or more R¹ monomers has a structure accordingto R^(1e):

In some embodiments, each R^(1e) group is independently selected from

In some embodiments, a mixture of the two stereoisomers are chosen. Insome embodiments, only the R stereoisomer of the monomer is chosen. Insome embodiments, only the S stereoisomer of this monomer is chosen.

Whenever any monomer herein does not indicate stereochemistry, anystereoisomer may be used. In some embodiments, a mixture of the twostereoisomers are chosen. In embodiments comprising a mixture ofstereoisomers, the ratio of R to S stereoisomer of the monomer in thepeptoid polymer can range from about 95:5 to about 90:10, from about90:10 to about 85:15, from about 85:15 to about 80:20, from about 80:20to about 75:25, from about 75:25 to about 70:30, from about 70:30 toabout 65:35, from about 65:35 to about 60:40, from about 60:40 to about55:45, from about 55:45 to about 50:50, from about 50:50 to about 45:55,from about 45:55 to about 40:60, from about 40:60 to about 35:65, fromabout 35:65 to about 30:70, from about 30:70 to about 25:75, from about25:75 to about 20:80, from about 20:80 to about 15:85, from about 15:85to about 10:90, or from about 10:90 to about 5:95. In some embodiments,only the R stereoisomer of the monomer is chosen. In some embodiments,only the S stereoisomer of the monomer is chosen.

Whenever a particular stereochemistry is shown with a wedge or a dashedline, the monomer is substantially free of other stereoisomers. In someembodiments, substantially free means at least 70% pure. In someembodiments, substantially free means at least 80% pure. In someembodiments, substantially free means at least 90% pure. In someembodiments, substantially free means at least 95% pure. In someembodiments, substantially free means at least 99% pure. In someembodiments, substantially free means at least 99.9% pure.

In some embodiments, each instance of Rl in the peptoid polymer isselected from the group consisting of:

In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more instances of R¹ in the peptoidpolymer are independently selected C₁₋₁₈ hydroxyalkyl groups (e.g.,independently selected C₁₋₆ hydroxyalkyl groups). In some embodiments,each instance of R¹ in the peptoid polymer is a C₁₋₁₈ hydroxyalkylgroup. In some embodiments, each instance of R¹ is a C₁₋₆ hydroxyalkylgroup. In some embodiments, each instance of R¹ is the same C₁₋₆hydroxyalkyl group. In some embodiments, each instance of R¹ is anhydroxyalkyl group where the length of the alkyl in each hydroxyalkylgroup is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 or more carbon atoms. In some embodiments thehydroxyalkyl group contains 1, 2, 3, 4, 5, 6, 7, or 8 hydroxysubstitutions. In some embodiments, each instance of R¹ is:

In some embodiments, each instance of R² is H. In some embodiments atleast one R² is a halogen.

In some embodiments, the sequence length of the peptoid polymer, n, isbetween 3 and 25. In some embodiments, the sequence length of thepeptoid polymer, n, is between 5 and 25. In some embodiments, thesequence length of the peptoid polymer, n, is between 8 and 50. In someembodiments, the sequence length of the peptoid polymer, n, is between 8and 25. In some embodiments, the sequence length of the peptoid polymer,n, is between 8 and 20. In some embodiments, the sequence length of thepeptoid polymer, n, can be between from about 10 to about 28, from about12 to about 26, from about 14 to about 24, from about 16 to about 22, orfrom about 18 to about 20. In some embodiments, the sequence length ofthe peptoid polymer, n, can be between from about 8 to about 50, fromabout 8 to about 45, from about 8 to about 40, from about 8 to about 35,from about 8 to about 30, from about 10 to about 25, from about 10 toabout 20, or from about 10 to about 15. In some embodiments, thesequence length of the peptoid polymer, n, can be 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13 ,14 , 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, or 50.

In some embodiments, X and Y are H, optionally substituted C₁₋₈alkylamino, —OH, —SH, carboxy, optionally substituted C₁₋₈ hydroxyalkyl,optionally substituted C₁₋₈ alkylamino, optionally substituted C₂₋₈alkylthio, optionally substituted C₁₋₈ carboxyalkyl, or halogen.

In some embodiments, X and Y of the peptoid polymer are taken togetherto form a covalent bond. The formation of a covalent bond between X andY results in a circularized form of the peptoid polymer in which theterminal NR¹ group and the terminal C═O group are linked, as shownbelow.

In some embodiments, the peptoid polymer consists of monomer unitsselected from the group of monomers set forth in Table 1. A person ofskill in the art will recognize that the bounds of this invention arenot limited to the monomers listed in Table 1, and that any usefulN-substituted substituent can be used as an N-substituted peptoidmonomer. In some embodiments, the N-substituted substituent on theN-substituted peptoid monomer is any of the side chains of the aminoacids alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid(Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine(Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met),asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser),threonine (Thr), valine (Val), tryptophan (Trp), or tyrosine (Tyr).

TABLE 1

  2-(sec-butylamino)acetic acid Nsb

  2-((2-hydroxypropyl)amino)acetic acid Nhp

  2-(isobutylamino)acetic acid Nib

  2-((2-hydroxyethyl)amino)acetic acid Nhe

  2-(butylamino)acetic acid Nbu

  2-((2,3-dihydroxypropyl)amino)acetic acid Ndp

  2-(propylamino)acetic acid Npr

  2-((1-hydroxypropan-2-yl)amino)acetic acid Nyp

  2-(isopropylamino)acetic acid Nip

  2-((1-(4-hydroxyphenyl)ethyl)amino)acetic acid Nep

  2-(methylamino)acetic acid Nme

  2-((1,3-dihydroxypropan-2-yl)amino)acetic acid Ndh

  2-((3-(2-oxopyrrolidin-1-yl)propyl)amino)acetic acid Nop

In some embodiments, the peptoid polymer is selected from the group ofpeptoid polymers set forth in Table 2, Table 3, Table 4, Table 5, Table6, Table 7, Table 8, or Table 9.

TABLE 2

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

TABLE 3

Compound 14

Compound 15

Compound 16

Compound 17

Compound 18

Compound 19

Compound 20

Compound 21

Compound 22

TABLE 4

Compound 23

Compound 24

TABLE 5

Compound 25

Compound 26

Compound 27

Compound 28

TABLE 6

Compound 29

Compound 30

Compound 31

Compound 32

Compound 33

Compound 34

TABLE 7

Compound 35

Compound 36

Compound 37

Compound 38

Compound 39

Compound 40

TABLE 8

Compound 41

Compound 42

Compound 43

Compound 44

Compound 45

Compound 46

Compound 47

Compound 48

Compound 49

TABLE 9

Compound 50

Compound 51

Compound 52

Compound 53

Compound 54

Compound 55

Compound 56

Compound 57

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 10 Nsb monomers, 1 Nhp monomerand 9 Nsb monomers, 2 Nhp monomers and 8 Nsb monomers, 3 Nhp monomersand 7 Nsb monomers, 4 Nhp monomers and 6 Nsb monomers, 5 Nhp monomersand 5 Nsb monomers, 6 Nhp monomers and 4 Nsb monomers, 7 Nhp monomersand 3 Nsb monomers, 8 Nhp monomers and 2 Nsb monomers, 9 Nhp monomersand 1 Nsb monomer, or 10 Nhp monomers.

In some embodiments, the peptoid polymer has the sequenceNhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp (SEQ ID NO:2), wherein X is H orC₁₋₈ acyl and Y is —OH or —NH₂ or C₁₋₈ alkyl. In some embodiments, thepeptoid polymer has the sequence Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb(SEQ ID NO:1), wherein X is H or C₁₋₈ acyl and Y is —OH or —NH₂ or C₁₋₈alkyl. In some embodiments, the peptoid polymer has the sequenceNsb-Nhp-Nhp-Nhp-Nsb-Nhp-Nhp-Nhp-Nsb-Nhp (SEQ ID NO:7), wherein X is H orC₁₋₈ acyl and Y is —OH or —NH₂ or C₁₋₈ alkyl. In some embodiments, thepeptoid polymer has the sequence Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb(SEQ ID NO:8), wherein X is H or C₁₋₈ acyl and Y is —OH or —NH₂ or C₁₋₈alkyl. In some embodiments, the peptoid polymer has the sequenceNsb-Nhp-Nhp-Nhp-Nhp-Nhp-Nsb-Nhp-Nhp-Nhp (SEQ ID NO:9), wherein X is H orC₁₋₈ acyl and Y is —OH or —NH₂ or C₁₋₈ alkyl. In some embodiments, Y isa secondary amine or a tertiary amine.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 10 Nme monomers, 1 Nhp monomerand 9 Nme monomers, 2 Nhp monomers and 8 Nme monomers, 3 Nhp monomersand 7 Nme monomers, 4 Nhp monomers and 6 Nme monomers, 5 Nhp monomersand 5 Nme monomers, 6 Nhp monomers and 4 Nme monomers, 7 Nhp monomersand 3 Nme monomers, and 8 Nhp monomers and 2 Nme monomers, or 9 Nhpmonomers and 1 Nme monomer.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 1 Nhe monomers and 9 Nsbmonomers, 2 Nhe monomers and 8 Nsb monomers, 3 Nhe monomers and 7 Nsbmonomers, 4 Nhe monomers and 6 Nsb monomers, 5 Nhe monomers and 5 Nsbmonomers, 6 Nhe monomers and 4 Nsb monomers, 7 Nhe monomers and 3 Nsbmonomers, 8 Nhe monomers and 2 Nsb monomers, 9 Nhe monomers and 1 Nsbmonomers, or 10 Nhe monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 10 Nbu monomers, 1 Nhp monomerand 9 Nbu monomers, 2 Nhp monomers and 8 Nbu monomers, 3 Nhp monomersand 7 Nbu monomers, 4 Nhp monomers and 6 Nbu monomers, 5 Nhp monomersand 5 Nbu monomers 6 Nhp monomers and 4 Nbu monomers, 7 Nhp monomers and3 Nbu monomers, 8 Nhp monomers and 2 Nbu monomers, or 9 Nhp monomers and1 Nbu monomer.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 10 Nib monomers, 1 Nhp monomerand 9 Nib monomers, 2 Nhp monomers and 8 Nib monomers, 3 Nhp monomersand 7 Nib monomers, 4 Nhp monomers and 6 Nib monomers, 5 Nhp monomersand 5 Nib monomers, 6 Nhp monomers and 4 Nib monomers, 7 Nhp monomersand 3 Nib monomers, 8 Nhp monomers and 2 Nib monomers, or 9 Nhp monomersand 1 Nib monomer.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 10 Npr monomers, 1 Nhp monomerand 9 Npr monomers, 2 Nhp monomers and 8 Npr monomers, 3 Nhp monomersand 7 Npr monomers, 4 Nhp monomers and 6 Npr monomers, 5 Nhp monomersand 5 Npr monomers, 6 Nhp monomers and 4 Npr monomers, 7 Nhp monomersand 3 Npr monomers, 8 Nhp monomers and 2 Npr monomers, or 9 Nhp monomersand 1 Npr monomer.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 10 Nip monomers, 1 Nhp monomerand 9 Nip monomers, 2 Nhp monomers and 8 Nip monomers, 3 Nhp monomersand 7 Nip monomers, 4 Nhp monomers and 6 Nip monomers, 5 Nhp monomersand 5 Nip monomers, 6 Nhp monomers and 4 Nip monomers, 7 Nhp monomersand 3 Nip monomers, 8 Nhp monomers and 2 Nip monomers , or 9 Nhpmonomers and 1 Nip monomer.

In some embodiments, the sequence length of the peptoid polymer, n, is14, and the peptoid polymer comprises: 6 Nhp monomers and 8 Nsbmonomers, 7 Nhp monomers and 7 Nsb monomers, 8 Nhp monomers and 6 Nsbmonomers, 10 Nhp monomers and 4 Nsb monomers, or 14 Nhp monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is14, and the peptoid polymer comprises: 6 Nhp monomers and 8 Nibmonomers, 7 Nhp monomers and 7 Nib monomers, 8 Nhp monomers and 6 Nibmonomers, 10 Nhp monomers and 4 Nib monomers, or 14 Nhp monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is16, and the peptoid polymer comprises: 5 Nhp monomers and 11 Nsbmonomers, 7 Nhp monomers and 9 Nsb monomers, 8 Nhp monomers and 8 Nsbmonomers, 10 Nhp monomers and 6 Nsb monomers, 12 Nhp monomers and 4 Nsbmonomers, or 16 Nhp monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is22, and the peptoid polymer comprises: 7 Nhp monomers and 15 Nsbmonomers, 10 Nhp monomers and 12 Nsb monomers, 11 Nhp monomers and 11Nsb monomers, 14 Nhp monomers and 8 Nsb monomers, 17 Nhp monomers and 5Nsb monomers, or 22 Nhp monomers.

In some embodiments, the peptoid polymer described herein forms ahelical structure. In some embodiments, the helical structure adopts astructure analogous to a polyproline helix. In certain instances, thepeptoid polymer forms a polyproline I helix. In certain other instances,the peptoid polymer forms a polyproline II helix. In some embodiments, ahelical structure is adopted when the peptoid polymer comprises at leastone N-Aryl side chain. In some embodiments, the N-Aryl side chain is aNep monomer.

In some embodiments, the peptoid polymer reduces or inhibits ice crystalformation at a temperature within about 0° C. to about −20° C. In otherembodiments, the peptoid polymer reduces or inhibits ice crystalformation at a temperature within about −20° C. to about −40° C. Incertain embodiments, the peptoid polymer reduces or inhibits ice crystalformation at about −20° C. In certain other embodiments, the peptoidpolymer reduces or inhibits ice crystal formation at a temperaturewithin about −40° C. to about −200° C. (e.g., about −196° C.).

In some embodiments, the peptoid polymer reduces or inhibits ice crystalformation at a temperature within about 0° C. to about −200° C., withinabout −10° C. to about −190° C., within about −20° C. to about −180° C.,within about −30° C. to about −170° C., within about −40° C. to about−160° C., within about −50° C. to about −150° C., within about −60° C.to about −140° C., within about −70° C. to about −140° C., within about−80° C. to about −130° C., within about −90° C. to about −120° C., orwithin about −100° C. to about −110° C.

In other embodiments, the peptoid polymer reduces or inhibits icecrystal formation at or about −10° C., at or about −15° C., at or about−25° C., at or about −30° C., at or about −35° C., at or about −40° C.,at or about −45° C., at or about −50° C., at or about −55° C., at orabout −60° C., at or about −65° C., at or about −70° C., at or about−75° C., at or about −80° C., at or about −85° C., at or about −90° C.,at or about −95° C., at or about −100° C., at or about −105° C., at orabout −110° C., at or about −115° C., at or about −120° C., at or about−125° C., at or about −130° C., at or about −135° C., at or about −140°C., at or about −145° C., at or about −150° C., at or about −155° C., ator about −160° C., at or about −165° C., at or about −170° C., at orabout −175° C., at or about −180° C., at or about −185° C., at or about−190° C., at or about −195° C., at or about −196° C., or at or about−200° C.

In some embodiments, the concentration of the peptoid polymer (e.g.,present in a composition, formulation, or product such as acryoprotectant solution, antifreeze solution, frozen food product, orcosmetic care product) is between about 100 nM and about 100 mM. Incertain embodiments, the concentration of the peptoid polymer (e.g.,present in a composition, formulation, or product such as acryoprotectant solution, antifreeze solution, frozen food product, orcosmetic care product) is between about 100 nM and about 250 nM, betweenabout 250 nM and about 500 nM, between about 500 nM and about 750 nM,between about 750 nM and about 1 μM, between about 1 μM and about 5 μM,between about 5 μM and about 25 μM, between about 25 μM and about 50 μM,between about 50 μM and about 100 μM, between about 100 μM and about 250μM, between about 250 μM and about 500 μM, between about 500 μM andabout 750 μM, between about 750 μM and about 1 mM, between about 1 mMand about 10 mM, between about 10 mM and about 50 mM, or between about50 mM and about 100 mM. In other embodiments, the concentration of thepeptoid polymer (e.g., present in a composition, formulation, or productsuch as a cryoprotectant solution, antifreeze solution, frozen foodproduct, or cosmetic care product) is about 100 nM, about 1 μM, about 10μM, about 100 μM, about 1 mM, about 10 mM, or about 100 mM. Inparticular embodiments, the concentration of the peptoid polymer isabout 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM.

B. Peptoid-Peptide Hybrids

In another aspect, the invention provides a peptoid-peptide hybrid. Insome embodiments, the peptoid-peptide hybrid comprises a peptoid polymerdescribed herein and one or more amino acids. The amino acids can benaturally-occurring amino acids or variants thereof. In someembodiments, the peptoid-peptide hybrid comprises between about 1 and 10amino acids (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).In other embodiments, the peptoid-peptide hybrid comprises between about10 and 100 amino acids (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids). In someembodiments, the peptoid-peptide hybrid comprises more than about 100amino acids. In other embodiments, the peptoid-peptide hybrid comprisesbetween 2 and 50 peptoid monomers (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 42, 44, 45, 46, 47,48, 49, or 50 peptoid monomers) and at least between about 1 and 100amino acids (e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 aminoacids).

The amino acids can be located at any position within the polymer,including at the N- and C-terminal ends and/or in between any of thepeptoid monomers. In instances where the peptoid-peptide hybridcomprises two or more amino acids, the amino acids may all becontiguous, or only a portion of them may be contiguous. Alternatively,all of the amino acids may be separated by one or more peptoid monomers.

In some embodiments, the amino acids are D-amino acids. In otherembodiments, the amino acids are L-amino acids. In some otherembodiments, the peptoid-peptide hybrid comprises a combination of D-and L-amino acids. In some embodiments, the one or more amino acids areselected from the group consisting of alanine, cysteine, aspartic acid,glutamic acid, phenylalanine, glycine, histidine, isoleucine, arginine,lysine, leucine, methionine, asparagine, proline, glutamine, serine,threonine, valine, tryptophan, tyrosine, and a combination thereof. Insome instances, the one or more amino acids are selected from the groupconsisting of isoleucine, threonine, alanine, and a combination thereof.

In some embodiments, one or more Nsb peptoid monomers in a peptoidpolymer are replaced with one or more isoleucine amino acid residues tocreate a peptoid-peptide hybrid. The one or more isoleucine amino acidscan be D-amino acids, L-amino acids, or a combination thereof. In otherembodiments, one or more Nhp peptoid monomers in a peptoid polymer arereplaced with one or more threonine amino acid residues to create apeptoid-peptide hybrid. The one or more threonine amino acids can beD-amino acids, L-amino acids, or a combination thereof. In some otherembodiments, one or more Nme peptoid monomers in a peptoid polymer arereplaced with one or more alanine amino acid residues to create apeptoid-peptide hybrid. The one or more alanine amino acids can beD-amino acids, L-amino acids, or a combination thereof.

In some embodiments, the peptoid-peptide hybrid comprises the sequence:

Nep-Nep-Xaa-Xaa-Xaa-Xaa-Nep-Nep-Nep-Nep-Nme-Nme (SEQ ID NO:3);

wherein the Xaa amino acid residues are independently selected aminoacids such as D-amino acids, L-amino acids, or a combination thereof. Asa non-limiting example, all instances of Xaa are Arg, Ala, Val, and/orSer amino acid residues.

In other embodiments, the peptoid-peptide hybrid comprises the sequence:

Nme-Nme-Xaa-Nme-Nme-Nme-Nme-Nhp-Nhp-Nsb-Xaa-Nme-Nme-Xaa-Nme-Nme-Nme (SEQID NO:4);

wherein the Xaa amino acid residues are independently selected aminoacids such as D-amino acids, L-amino acids, or a combination thereof. Asa non-limiting example, all instances of Xaa are Arg, Ala, Val, and/orIle amino acid residues.

In yet other embodiments, the peptoid-peptide hybrid comprises thesequence:

Nme-Nme-Xaa-Nme-Nme-Nme-Nme-Nme-Nme-Nme-Xaa-Xaa (SEQ ID NO:5);

wherein the Xaa amino acid residues are independently selected aminoacids such as D-amino acids, L-amino acids, or a combination thereof. Asa non-limiting example, all instances of Xaa are Arg, Ala, Val, and/orLeu amino acid residues.

In some embodiments, the peptoid-peptide hybrid comprises the sequence:

Arg-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb (SEQ ID NO:6);

wherein the Arg amino acid residue is a D-amino acid or an L-amino acid.In some embodiments, the peptoid-peptide hybrid comprises the structureset forth in Table 10.

TABLE 10

Compound 58

C. Methods of Synthesis

In another aspect, the invention herein provides a method ofsynthesizing a peptoid polymer or a peptoid-peptide hybrid. The peptoidpolymers and peptoid-peptide hybrids of the invention can be preparedfrom readily available starting materials using the general methods andprocedures described herein. It will be appreciated that where typicalor preferred process conditions (i.e., reaction temperatures, times,mole ratios of reactants, solvents, pressures, etc.) are given, otherprocess conditions can also be used unless otherwise stated. Optimumreaction conditions may vary with the particular reactants or solventused, but such conditions can be determined by one skilled in the art byroutine optimization procedures.

The peptoid polymers and peptoid-peptide hybrids of the invention may beprepared from known or commercially available starting materials andreagents by one skilled in the art of organic synthesis. Solvents andreagents are purchased from commercial sources and used without furtherpurification.

In some embodiments, the submonomer approach (FIG. 1) is used forpeptoid synthesis, where each N-substituted glycine monomer is assembledfrom two readily available “submonomers.” The synthesis of oligomericpeptoids is based on the robust chemistry of standard solid-phasemethods, analogous to peptide synthesis. Each cycle of monomer additionconsists of two steps, an acylation step and a nucleophilic displacementstep. In some embodiments, solid-phase assembly eliminates the need forN-protected monomers because there are no reactive side chainfunctionalities that need to be protected. One of skill in the art willrecognize there are many solid-phase synthesis methods, includingautomated, robotic synthesizers. In some embodiments, the synthesizerused is the Symphony® X Multiplex Peptide Synthesizer made by ProteinTechnologies, Inc. In some embodiments, the synthesizer used is theOverture Peptide Synthesizer made by Protein Technologies, Inc. In otherembodiments, the peptoids are synthesized manually using traditionalorganic chemistry methods known in the art. By providing the appropriateamino acids in place of peptoid monomers at the appropriate times duringsynthesis, the same techniques or techniques similar to those describedabove can be applied to the synthesis of peptoid-peptide oligomers.

As a non-limiting example, peptoid polymers can be synthesized on 100 mgof Rink amide resin (NovaBiochem; 0.49 mmol/g). Rink amide resin (100mg) can be washed twice in 1.5 mL of DCM, followed by swelling in 1.5 mLof DMF. The swelling step can be performed twice. The Fmoc protectinggroup can be removed from the resin by addition of 20% piperidine/DMF.The mixture can be agitated for 10 minutes, drained, and the piperidinetreatment repeated, followed by extensive washes with DMF (five timeswith 1.5 mL). The first monomer can be added manually by reacting 37 mgof bromoacetic acid (0.27 mmol; Sigma-Aldrich) and 189 μL of DIEA (1.08mmol; Chem Impex International) in 2 mL of DCM on a shaker platform for30 minutes at room temperature, followed by extensive washes with DCM(five times with 2 mL) and DMF (five times with 2 mL). Bromoacylatedresin can be incubated with 2 mL of 1 M amine submonomer in DMF on ashaker platform for 30 minutes at room temperature, followed byextensive washes with DMF (five times with 2 mL). After initial manualloading of bromoacetic acid, the first submonomer displacement step andall subsequent bromo acetylation and amine displacement steps can beperformed by a robotic synthesizer until the desired oligomer length isobtained. The automated bromoacetylation step can be performed by adding1660 μL of 1.2 M bromoacetic acid in DMF and 400 μL of DIC (Chem ImpexInternational). The mixture can be agitated for 20 min, drained, andwashed with DMF (three times with 2 mL). Next, 2 mL of a 1 M solution ofsubmonomer (2 mmol) in DMF can be added to introduce the side chain bynucleophilic displacement of bromide. The mixture can be agitated for 20min, drained, washed with DMF (three times with 2 mL) and washed withDCM (three times with 2 mL). The peptoid-resin can be cleaved in 2 mL of20% HFIP (Alfa Aesar) in DCM (v/v) at room temperature. The cleavage canbe conducted in a glass tube with constant agitation for 30 minutes.HFIP/DCM can be evaporated under a stream of nitrogen gas. The finalproduct can be dissolved in 5 mL of 50% ACN in HPLC grade H₂O andfiltered with a 0.5 pm stainless steel fritted syringe tip filter(Upchurch Scientific). Peptoid oligomers can be analyzed on a C₁₈reversed-phase analytical HPLC column at room temperature (PeekeScientific, 5 pm, 120 Å, 2.0×50 mm) using a Beckman Coulter System Goldinstrument. A linear gradient of 5-95% acetonitrile/water (0.1% TFA,Acros Organics) over 20 min can be used with a flow rate of 0.7 mL/min.In order to remove any traces of HFIP in the sample solution, linearprecursors dissolved in 50% ACN/H₂O can be freeze-dried overnight.

Peptoid polymers and peptoid-peptide hybrids can be analyzed byelectrospray ionization (ESI) mass spectrometry. Generally, 0.5-2 mL of1-5 μM of peptoid polymer or peptoid-peptide hybrid to be analyzed isprepared in a 50% deionized H₂O/50% HPLC grade ACN with 1% of an organicacid such as trifluoroacetic acid. Prepared samples are ionized bybombardment with electrons causing the molecules to break into chargedfragments. The ions are then separated according to their mass-to-chargeratio by accelerating the fragments and exposing them to an electricalor magnetic field. The ions are detected by a mechanism capable ofdetecting charged particles, such as an electron multiplier. Peptoidsand peptoid-peptide hybrids are identified by correlating masses to theidentified masses or through a characteristic fragmentation pattern.

D. Methods of Use

In some aspects, the present invention provides a cryoprotectantsolution. In some embodiments, the cryoprotectant solution comprises apeptoid polymer described herein, a peptoid-peptide hybrid describedherein, or a combination thereof. In other embodiments, thecryoprotectant solution further comprises a compound selected from thegroup consisting of an ionic species, a penetrating cryoprotectant, anon-penetrating cryoprotectant, an antioxidant, a cell membranestabilizing compound, an aquaporin or other channel forming compound, analcohol, a sugar, a sugar derivative, a nonionic surfactant, a protein,dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), Ficoll®,polyvinylpyrrolidone, polyvinyl alcohol, hyaluronan, formamide, anatural or synthetic hydrogel, and a combination thereof. In particularembodiments, the penetrating cryoprotectant penetrates the cell membraneand reduces the intracellular water concentration, thereby reducing theamount of ice formed at any temperature. In other particularembodiments, the non-penetrating cryoprotectant induces changes incolloidal osmotic pressure and modifies cell membrane associations withextracellular water by induced ionic interaction.

In some instances, the cryoprotectant solution further comprises analcohol that is selected from the group consisting of propylene glycol,ethylene glycol, glycerol, methanol, butylene glycol, adonitol, ethanol,trimethylene glycol, diethylene glycol, polyethylene oxide, erythritol,sorbitol, xythyritol, polypropylene glycol, 2-methyl-2,4-pentanediol(MPD), mannitol, inositol, dithioritol, 1,2-propanediol, and acombination thereof.

In other instances, the cryoprotectant solution further comprises asugar that is selected from the group consisting of a monosaccharide, adisaccharide, a polysaccharide, and a combination thereof. In particularinstances, the sugar is selected from the group consisting of glucose,3-O-Methyl-D-glucopyranose, galactose, arabinose, fructose, xylose,mannose, sucrose, trehalose, lactose, maltose, raffinose, dextran, and acombination thereof.

In other instances, the cryoprotectant solution further comprises PEG ora plurality of different PEG compounds. In some other instances, atleast one of the PEG compounds has an average molecular weight less thanabout 1,000 g/mol (e.g., less than about 1,000 950, 900, 850, 800, 750,700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100g/mol). In particular instances, a least one of the PEG compounds has anaverage molecular weight between about 200 and 400 g/mol (e.g., about200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, or 400 g/mol). In some instances, thecryoprotectant solution comprises PEG or a plurality of PEG compoundsselected from the group consisting of PEG 200, PEG 300, PEG 400, and acombination thereof.

In other instances, the cryoprotectant solution further comprises aprotein that is selected from the group consisting of egg albumin,bovine serum albumin, human serum albumin, gelatin, and a combinationthereof. In still other instances, the cryoprotectant solution furthercomprises a natural or synthetic hydrogel, wherein the natural orsynthetic hydrogel comprises chitosan, hyaluronic acid, or a combinationthereof.

Non-limiting examples of various properties of the cryoprotectantsolution such as effective concentration, viscosity, water solubility,and/or membrane permeability can be assessed using a model cell ortissue including, but not limited to, stem cells, liver tissue orhepatocytes, kidney, intestine, heart, pancreas, bone marrow, organoids,and other biological tissues for cryopreservation.

In some embodiments, the cryoprotectant solution reduces or inhibits icecrystal formation at a temperature within about 0° C. to about −20° C.In other embodiments, the cryoprotectant solution reduces or inhibitsice crystal formation at a temperature within about −20° C. to about−40° C. In certain embodiments, the cryoprotectant solution reduces orinhibits ice crystal formation at about −20° C. In certain otherembodiments, the cryoprotectant solution reduces or inhibits ice crystalformation at a temperature within about −40° C. to about −200° C. (e.g.,about −196° C.).

In some embodiments, the cryoprotectant solution reduces or inhibits icecrystal formation at a temperature within about 0° C. to about −200° C.,within about −10° C. to about −190° C., within about −20° C. to about−180° C., within about −30° C. to about −170° C., within about −40° C.to about −160° C., within about −50° C. to about −150° C., within about−60° C. to about −140° C., within about −70° C. to about −140° C.,within about −80° C. to about −130° C., within about −90° C. to about−120° C., or within about −100° C. to about −110° C.

In other embodiments, the cryoprotectant solution reduces or inhibitsice crystal formation at or about −10° C., at or about −15° C., at orabout −25° C., at or about −30° C., at or about −35° C., at or about−40° C., at or about −45° C., at or about −50° C., at or about −55° C.,at or about −60° C., at or about −65° C., at or about −70° C., at orabout −75° C., at or about −80° C., at or about −85° C., at or about−90° C., at or about −95° C., at or about −100° C., at or about −105°C., at or about −110° C., at or about −115° C., at or about −120° C., ator about −125° C., at or about −130° C., at or about −135° C., at orabout −140° C., at or about −145° C., at or about −150° C., at or about−155° C., at or about −160° C., at or about −165° C., at or about −170°C., at or about −175° C., at or about −180° C., at or about −185° C., ator about −190° C., at or about −195° C., at or about −196° C., or at orabout −200° C.

In some embodiments, the concentration of the peptoid polymer and/orpeptoid-peptide hybrid in the cryoprotectant solution is between about100 nM and about 100 mM. In some embodiments, the concentration ofpeptoid polymer and/or peptoid-peptide hybrid in the cryoprotectantsolution is between about 100 nM and about 250 nM, between about 250 nMand about 500 nM, between about 500 nM and about 750 nM, between about750 nM and about 1 μM, between about 1 μM and about 5 μM, between about5 μM and about 25 μM, between about 25 μM and about 50 μM, between about50 μM and about 100 μM, between about 100 μM and about 250 μM, betweenabout 250 μM and about 500 μM, between about 500 μM and about 750 μM,between about 750 μM and about 1 mM, between about 1 mM and about 10 mM,between about 10 mM and about 50 mM, or between about 50 mM and about100 mM. In some embodiments, the concentration of the peptoid polymerand/or peptoid-peptide hybrid in the cryoprotectant solution is about100 nM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, about 10 mM,or about 100 mM. In particular embodiments, the concentration of thepeptoid polymer and/or peptoid-peptide hybrid in the cryoprotectantsolution is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM.

In other aspects, provided herein is a method for preserving abiological sample. In particular embodiments, the biological samplepossesses cellular composition. In some embodiments, the biologicalsample is a tissue. In other embodiments, the biological sample is anorgan. In still other embodiments, the biological sample is a cell. Inparticular embodiments, the biological sample comprises one or moretissues, organs, or cells, or a combination thereof. In someembodiments, the method comprises contacting the biological sample witha peptoid polymer described herein, a peptoid-peptide hybrid describedherein, a cryoprotectant solution described herein, or a combinationthereof. In some instances, when a combination of compositions orsolutions is used, contacting the biological sample with thecompositions or solutions can be accomplished in multiple steps. As anon-limiting example, a biological sample can first be contacted with apeptoid polymer described herein, and then at a later point thebiological sample can be contacted with a cryoprotection solutiondescribed herein.

In particular instances, the tissue is a bioengineered tissue. In someinstances, the biological sample is selected from the group consistingof heart, liver, lung, kidney, pancreas, intestine, thymus, cornea,nerve cells, blood platelets, sperm cells, oocytes, embryonic cells,stem cells, bone cells, and a combination thereof.

Cryoprotection of biological samples is useful for any number ofpurposes. Non-limiting examples include organoid preservation, stem cellpreservation (e.g., hematopoietic stem cells, embryonic stem (ES) cells,pluripotent stem cells (PSCs), and induced pluripotent stem cells(iPSCs)), preservation of adult cells and cell lines (e.g., lymphocytes,granulocytes, immune system cells, bone cells), preservation of embryos,sperm, and oocytes, tissue preservation, and organ preservation.Preservation of tissues, organs, and other biological samples andstructures is especially useful, for example, in the field of organtransplantation. Other useful applications of the present invention tobiological sample cryoprotection will readily be known to one of skillin the art.

In yet other aspects, provided herein is a method for preserving one ormore biological macromolecules. Said biological macromolecules can benaturally or unnaturally occurring. Non-limiting examples of biologicalmacromolecules that are suitable for cryoprotection by compositions andmethods of the present invention include nucleic acids (e.g., DNA, RNA),amino acids, proteins, peptides, lipids, and composite structures (e.g.,liposomes). In some embodiments, the method comprises contacting thebiological macromolecule with a peptoid polymer described herein, apeptoid-peptide hybrid described herein, a cryoprotectant solutiondescribed herein, or a combination thereof. In some instances, thebiological macromolecule is an isolated protein. In particularinstances, the isolated protein is a protease protein. In someinstances, when a combination of compositions or solutions is used,contacting the one or more biological macromolecules with thecompositions or solutions can be accomplished in multiple steps. As anon-limiting example, the one or more biological macromolecules canfirst be contacted with a peptoid polymer described herein, and then ata later point the biological sample can be contacted with acryoprotection solution described herein.

Cryoprotection of biological macromolecules using compositions andmethods of the present invention is useful for any number of purposes.Non-limiting examples of such purposes include the preservation of DNA(e.g., genomic DNA) and RNA samples, the preservation of stem cellgrowth factors, and the preservation of antibodies. Other usefulpurposes and applications appropriate for compositions and methods ofthe present invention will be readily known by one of skill in the art.

In particular embodiments, the isolated protein has been crystallized.Crystal cryoprotection has become an essential tool in the repertoire ofcrystallographic methods for studying biological macromolecules (e.g.,proteins and peptides). In many cases, cryoprotection and subsequentdata collection at cryogenic temperature are essential for obtaining acomplete data set by overcoming the problem of radiation damage from thex-ray beam line. Moreover, cryomethods allow crystallographers to workwith small crystals, and such methods have become an ideal method toperform long term storage of the crystals without losing diffractionquality. Cryoprotectants provide a means to protect macromolecularcrystals from the damaging effects of ice formation during thecryocooling process. Cryoprotection usually involves immersing thecrystal in a solution that forms an amorphous glass (i.e.,vitrification) while being flash cooled in liquid nitrogen. The idealcryoprotectants for crystallography should be hypereffective (i.e., thecryoprotectants achieve an effective result at a low concentration).Currently available cryoprotectants are not hypereffective. Therefore,if the cryoprotectant concentration is too low, crystalline ice willform during the experiment which leads to background interference. Ifthe cryoprotectant concentration is too high, the immediate melting downof the crystal structure can result from beam energy, resulting in lowquality data affecting subsequent structure analysis. For example,current state of the art cryoprotectant solutions used in x-raycrystallography applications require the use of 20% ethylene glycol toprevent ice crystal formation at crystalized protein storagetemperatures. During x-ray data collection, the ethylene glycol heatsand dissolves the crystals preventing further data collection. Foradditional information, see, e.g., Garman et al. J. Appl. Cryst. 30:211(1997).

In some embodiments, the peptoid polymer, peptoid-peptide hybrid, orcryoprotectant solution described herein, or a combination thereof,decreases crystal dissolving during x-ray data collection. In someembodiments, the peptoid polymer, peptoid-peptide hybrid, cryoprotectantsolution described herein, or a combination thereof, lowers backgroundscattering.

Biological samples and macromolecules that are suitable forcryoprotection according to the compositions and methods of the presentinvention can come from any biological kingdom (e.g., Animalia(including but not limited to humans and livestock animals), Plantae,Fungi (including but not limited to mushrooms), Protista,Archaea/Archaeabacteria, and Bacteria/Eubacteria).

In another aspect, the present invention provides a cosmetic careproduct. In some embodiments, the cosmetic care product comprises apeptoid polymer described herein, a peptoid-peptide hybrid describedherein, a cryoprotection solution described herein, or a combinationthereof. In some embodiments, the cosmetic care product is a skin careproduct. In some embodiments the skin care product is topically applied.Typical formulations for topical products include creams, serums,ointments, sprays, lotions, and patches.

In another aspect, the present invention provides an antifreeze productsuch as a deicing or ice inhibiting product. In some embodiments, theantifreeze product comprises a peptoid polymer described herein, apeptoid-peptide hybrid described herein, a cryoprotection solutiondescribed herein, or a combination thereof. In some embodiments, theantifreeze product is used to prevent, inhibit, or delay the formationof ice on objects including, but not limited to, general mechanical andelectrical equipment. In some embodiments, the antifreeze productprevents, inhibits, or delays the formation of ice on aircraft or partsthereof, drones, automobiles or parts thereof, including car engines,gear systems, brake systems, windows, sprinkler systems, gas pipelines,or electrical cables, including powerlines. In other instances, theantifreeze product acts as a kinetic hydrate inhibitor. In someembodiments the antifreeze product further comprises ethylene glycol,methanol, propylene glycol, glycerol, or combinations thereof.

In another aspect, the present invention provides a frozen food product.In some embodiments the frozen food product comprises a peptoid polymerdescribed herein, a peptoid-peptide hybrid described herein, acryoprotection solution described herein, or a combination thereof. Insome embodiments, the frozen food product is selected from the groupconsisting of ice cream, yogurt, seafood, fruit, and meat products. Insome embodiments the frozen food product further comprises propyleneglycol.

E. Cryopreservation Protocols

The compositions and methods described herein are suitable for use inany number of cryopreservation protocols. As a non-limiting example,compositions and methods of the present invention are useful forcryopreservation during supercooling to high sub-zero temperatures(e.g., 0° C. to −20° C.). In the field of organ transplantation, organsare typically cooled on ice (e.g., to 0-4° C.), which limits thetransplantation window to about ten hours. By using ex vivo machineperfusion with cryoprotectants containing standard small molecule CPAs,it has been possible to preserve organs for up to 96 hours at atemperature of −6° C. While it is desirable to further reduce thecryopreservation temperature below −6° C., which would extend thepossible cryopreservation time, it has not been possible to do sobecause the high concentrations of standard CPAs necessary to furtherreduce the temperature result in irreversible organ damage owing toCPA-related toxicity. For more information, see, e.g., Uygun K, et. al.Nat. Protoc. 10(3):484-94 (2015). Employing ex vivo perfusion methods orotherwise contacting biological samples (e.g., organs and tissues) ormacromolecules with peptoid polymers, peptoid-peptide hybrids, and/orcryoprotectant solutions described herein is useful for supercooling tohigh sub-zero temperatures, allowing cryopreservation for longer periodsof time and at lower temperatures than is currently feasible. Othersuitable applications of the present invention to high sub-zerotemperature supercooling will readily be known to one of skill in theart.

As another non-limiting example, compositions and methods of the presentinvention are useful for cryopreservation during freezing protocols(e.g., −20° C. to −196° C.). Freezing protocols are typically performedat a controlled rate (sometimes referred to as slow freezing) during atleast part of the temperature reduction. For example, a biologicalsample or macromolecule can be contacted with a peptoid polymer,peptoid-peptide hybrid, and/or cryoprotectant solution described herein,and the temperature can be reduced at a controlled rate (e.g., loweredat a rate of 1° C. per minute) until the desired temperature is reached.Alternatively, the temperature can be reduced at a controlled rate untila desired temperature is reached (e.g., between −80° C. and −180° C.),and then the sample or macromolecule can be flash frozen (e.g., byimmersing the sample or macromolecule in liquid nitrogen or placing thesample or macromolecule above liquid nitrogen). The peptoid polymer,peptoid-peptide hybrid, or cryoprotectant solution can be contacted withthe sample or macromolecule being cryopreserved at any point during theprotocol, as long as it is before the formation of ice crystals thatdamage the sample or macromolecule being preserved.

As yet another non-limiting example, compositions and methods of thepresent invention are useful for cryogenic freezing protocols (e.g.,−90° C. to −196° C.). For example, a biological sample or macromoleculecan be contacted with a peptoid polymer, peptoid-peptide hybrid, orcryoprotectant solution described herein, then plunged into liquidnitrogen or a stream of liquid nitrogen vapor in order to quickly freezethe sample without the formation of ice crystals. No ice lattice existsand so the water within the sample or macromolecule is in an amorphousor glass-like state. Therefore, damaging ice is not formed.

One of skill in the art will readily appreciate that the concentrationsand compositions of the peptoid polymers, peptoid-peptide hybrids, andcryoprotectant solutions described herein can be modified depending onthe particular biological sample and/or macromolecule beingcryopreserved and the particular cryopreservation protocol beingemployed.

F. Methods of Screening

In a related aspect, the present invention provides methods forscreening peptoid polymers, peptoid-peptide hybrids, and/orcryoprotectant solutions for activity.

In one embodiment, the peptoid polymer, peptoid-peptide hybrid, and/orcryoprotectant solution is screened for lowering the freezing point ofwater using a polarized light microscope to detect ice crystalformation. Polarized light microscopy is an optical microscopy techniquethat uses polarized light as the light source. Image contrast arisesfrom the interaction of plane-polarized light with a birefringent (ordoubly-refracting) species to produce two individual wave componentsthat are each polarized in mutually perpendicular planes. The velocitiesof these components, which are termed the ordinary and the extraordinarywavefronts, are different and vary with the propagation directionthrough the specimen. After exiting the specimen, the light componentsbecome out of phase, but are recombined with constructive anddestructive interference when they pass through the analyzer. Thisinterference creates a detectable contrast in the sample. Ice crystalformation is easily detected using this technique because ice crystalsare birefringent species. In a standard experiment, samples comprisingthe peptoid polymer, peptoid-peptide hybrid, and/or cryoprotectantsolution are cooled to a desired temperature for a desired amount oftime. One or more samples, while at the desired temperature, are placedunder the polarized light microscope and visually inspected forformation of ice crystals.

In one embodiment, the peptoid polymer, peptoid-peptide hybrid, and/orcryoprotectant solution is screened for lowering the freezing point ofan aqueous solution using differential scanning calorimetry toquantitate thermal hysteresis activity. Differential scanningcalorimetry is a thermoanalytical technique in which the difference inthe amount of heat required to increase the temperature of a sample andreference is measured as a function of temperature. When a physicaltransformation such as phase transition occurs, more or less heat willneed to flow to the sample than the reference to maintain both at thesame temperature. The difference in temperature between the phasetransition of the reference and the sample reports on the sample'sability to reduce or inhibit ice crystal formation at sub 0° C.temperatures. In a standard experiment, a sample comprising the peptoidpolymer, peptoid-peptide hybrid, and/or cryoprotectant solution iscompared to a reference that lacks the peptoid polymer, peptoid-peptidehybrid, and/or cryoprotectant solution.

G. Cell Viability Assays to Test for Activity

In a related aspect, the present invention provides cell viabilityassays to test for the ability of the peptoid polymer, peptoid-peptidehybrid, and/or cryoprotectant solution to maintain cell viability atreduced temperatures.

In some embodiments, cell viability is tested using the alamarBlue® CellViability Assay Protocol provided by Thermo Fisher Scientific, Inc.Briefly, alamarBlue® is the trade name of resazurin(7-Hydroxy-3H-phenoxazin-3-one 10-oxide) which is a non-toxic cellpermeable compound that is blue in color and virtually non-fluorescent.Upon entering cells, resazurin is reduced to resorufin, a compound thatis red in color and highly fluorescent. Viable cells continuouslyconvert resazurin to resorufin, increasing the overall fluorescence andcolor of the media surrounding cells. Non-viable cells do not convertresazurin to resorufin, thus the overall fluorescence and color of themedia surrounding the cells is an indication of the relative amount ofviable cells in the sample. In a standard experiment, cells and thepeptoid polymer, peptoid-peptide hybrid, and/or cryoprotectant solutionare mixed in any suitable container. The mixture is then cooled to thedesired sub 0° C. temperature and held for the desired amount of time.Cells are then returned to ambient temperatures and the almarBlue®reagent is added, incubated, and measured following the Thermo Fisherprotocol. Typically, direct readout of cell viability is determined bymeasuring the relative fluorescence of the samples at the wavelengthsλ_(Ex)˜560 nm/λ_(Em)˜590 nm.

In some embodiments, cell viability is tested using the LIVE/DEAD®Viability/Cytotoxicity Kit, for mammal cells provided by Thermo FisherScientific, Inc. This kit uses two indicator molecules: calcein AM andEthidium homodoimer-1 (EthD-1). Live cells are distinguished by thepresence of ubiquitous intracellular esterase activity, determined bythe enzymatic conversion of the virtually nonfluorescent cell-permeantcalcein AM to the intensely fluorescent calcein. The polyanionic dyecalcein is well retained within live cells, producing an intense uniformgreen fluorescence in live cells (λ_(Ex)˜495 nm/λ_(Em)˜515 nm).Conversely, EthD-1 enters cells with damaged membranes and undergoes a40-fold enhancement of fluorescence upon binding to nucleic acids,thereby producing a bright red fluorescence in dead cells (λ_(Ex)˜495nm/λ_(Em)˜635 nm). Notably, EthD-1 is excluded by the intact plasmamembrane of live cells, so the determination of live and dead cells iseasily distinguishable. Calcein and EthD-1 can be viewed simultaneouslywith a conventional fluorescein longpass filter. Alternatively, thefluorescence from these dyes may also be observed separately; calceincan be viewed with a standard fluorescein bandpass filter, and EthD-1can be viewed with filters for propidium iodide or Texas Red® dye. In astandard experiment, cells and the peptoid polymer, peptoid-peptidehybrid, and/or cryoprotectant solution are mixed in any suitablecontainer. The mixture is then cooled to the desired sub 0° C.temperature, held at that temperature for the desired amount of time,and then returned to ambient temperatures. Subsequent steps involvingthe addition of the calcein AM and EthD-1 reagents and measuring theassay results are performed as described in the Thermo Fisher protocol.Typically, direct readout of cell viability is determined by measuringthe relative fluorescence at the above indicated wavelengths for bothreagents.

In some embodiments, cell viability is tested using the MTT assay. TheMTT assay is a colorimetric cell viability and proliferation assay thatrelies upon the reduction of yellow tetrazolium MTT(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) to theinsoluble formazan, which has a purple color. Tetrazolium dye reductionis dependent on NAD(P)H-dependent oxidoreductase enzymes, primarilylocated in the cytosolic compartment of metabolically active cells. TheMTT assay is available, for example, from ATCC (www.atcc.org) orSigma-Aldrich (www.sigmaaldrich.com). In a standard experiment, cellsand the peptoid polymer, peptoid-peptide hybrid, and/or cryoprotectantsolution are mixed in any suitable container. The mixture is then cooledto the desired sub 0° C. temperature and held for the desired amount oftime. Cells are then returned to ambient temperatures and the MTTreagent is added, incubated, and measured following the ATCC orSigma-Aldrich protocol. Typically, absorbance of converted dye ismeasured at a wavelength of 570 nm with background subtraction at630-690 nm.

IV. EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1 Peptoid-Mediated Inhibition of Ice Crystal Formation

This example illustrates the ice crystal inhibition properties ofN-substituted peptoid polymers and peptoid-peptide hybrids at sub 0° C.temperatures.

Capillary Tube Assays

In this experiment, four water-based samples were prepared in capillarytubes containing MilliQ purified water. One sample contained only water,and another sample contained 160 mM ethylene glycol (EG). The other twosamples each contained a peptoid polymer at 9 mM. One of the peptoidpolymer samples contained the peptoid polymer called “Compound 1,” whilethe other sample contained the peptoid polymer called “Compound 10.” Thesequences of the peptoid polymers are as follows:

Compound 1: (SEQ ID NO: 1) Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb;Compound 10: (SEQ ID NO: 2) Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp.

The chemical structures for these compounds are provided in Table 2.

After sample preparation, all samples were slow cooled and incubated at−20° C. on a Peltier cooled plate. After one hour, samples were removedand immediately photographed using a digital camera attached to a 180×Stereo Zoom microscope (FIG. 2A). The water and EG samples showedsignificant ice crystal formation, although the EG sample showed lessice formation than the water-only sample. In contrast, neither of thesamples containing the peptoid polymer compounds exhibited significantice crystal formation. Normalized data is presented in FIG. 2B. Of note,the EG sample, containing a CPA concentration that was about 18 timeshigher than the peptoid sample concentrations, still exhibitedsignificant ice formation whereas the peptoid samples did not.

Crystallographic X-Ray Diffraction Assays

In order to increase the throughput of library analysis, acrystallographic x-ray diffraction (XRD) technique was used to evaluateice crystal formation. For these experiments, the compounds named“Compound 2,” “Compound 8,” “Compound 10,” “Compound 11,” “Compound 12,”“Compound 13,” and “Compound 58” were tested. Compounds 2, 8, 10, 11,12, and 13 are peptoid polymers, the structures of which are provided inTable 2. Compound 58 is a peptoid-peptide hybrid, the structure of whichis provided in Table 10. Compound 58 is similar to Compound 12, exceptthat an arginine amino acid has been appended to the N-terminal end.

For these experiments, EG concentrations between 15% and 30% (v/v) wereused. Typically, EG, DMSO, and other cryoprotectants are used during XRDsample analysis at concentrations of 35-40% (v/v) to vitrify solutionsand avoid diffraction interference from ice crystals. Concentrations of1 and 5 mg/mL of the peptoid and peptoid-peptide compounds were used.FIGS. 3A, 3B, and 3C illustrate exemplary XRD data under conditions ofcomplete vitrification, partial vitrification with the presence of cubicice, and freezing (cubic ice crystals), respectively. XRD data forCompounds 8, 10, 11, 12, 13, and 58 is provided in FIGS. 4A-4G and FIGS.5A-5G. FIG. 3D provides ice rings scores for a variety of EGconcentrations and two concentrations of Compounds 2, 8, and 12.

Several mixtures of the testing solution sample sets showed a stronganti-icing effect. FIG. 3D shows the experimental results of somepeptoid polymer solutions compared to EG. “IceRing1” and “IceRing2”refer to ice formation scores, which range between 0 (no ice formation)and 15 (large ice formation). Compounds 2, 12, and 8 and otherssignificantly reduced necessary EG concentrations while preventing iceformation.

The sample containing Compound 12 at a concentration of 5 mg/mL (0.5%(w/v)) and EG at a concentration of 17.5% (v/v) in water was ice-freeafter flash freezing. This particular mixture was found to completelyeliminate all ice formation over multiple trials of flash freezing in astream of liquid nitrogen vapor (FIG. 3A), and vastly outperformed astandard solution of 30% EG (FIG. 3B). In the figures, black spots andrings represent ice crystals. In comparison to EG at the same molarconcentration, this anti-icing effect is 500 times stronger and, withoutbeing bound by any particular theory, suggests a non-colligativemechanism for anti-icing, which is the mechanism used by naturalantifreeze proteins.

Larger Volume Assays

In order to test the usefulness of compositions of the present inventionat larger scales, experiments were performed using solution volumes thatare similar to volumes used for standard egg and stem cell preservation.For these experiments, two samples, one containing 22.5% EG and bufferonly, and another containing 22.5% EG and 5 mg/ml (0.5% w/v) of Compound12 and buffer, were flash frozen in liquid nitrogen. As shown in FIG.6A, the Compound 12 solution showed complete vitrification with no iceformation immediately after removal from liquid nitrogen, while thecontrol solution had clearly been frozen, yielding a mass of white icecrystals. The rewarming of the solutions in a 37° C. water bath led toan unexpected and beneficial result. The Compound 12 solution bypasseddevitrification in less than 2 seconds upon rewarming (FIG. 6B, right),whereas chunks of ice were seen floating in the control sample (FIG. 6B,left) after 20 seconds. Condensation was seen on each of the tubesbecause the tubes were actually still much below room temperature. Thisresult shows that Compound 12 acts as an active de-icer during thawing.

Furthermore, after leaving the 100 μL samples in a −20° C. freezerovernight, the Compound 12 solution was found to be unfrozen (FIG. 6C,right). This result shows that compositions of the present inventionprovide the ability to preserve samples at below 0° C. temperatures forlong periods of time without any ice formation. Furthermore, theseexperiments show that ice-free conditions can be reached withhypothermic cryopreservation, or by the supercooling method, at −20° C.as well as near vitrification to −80° C. by incorporating compounds ofthe present invention to significantly reduce the critical concentrationof penetrating CPAs and mitigate cryopreservation toxicity.

As shown here, a formulation of Compound 12 was found to prevent iceformation during vitrification in sub-milliliter volumes. In fact, thesolutions were able to remain completely unfrozen at −20° C. and werealso able to vitrify when flash frozen at −196° C. Currently, standardhuman egg cell preservation techniques for in vitro fertilization arelimited to solution volumes of less than 5 uL (often 0.5 to 2.5 μL)while using 50% or greater cryoprotectant concentrations. Thus, Compound12 was able to prevent ice formation in a practical volume, withexceedingly less cryoprotectant, which makes it useful, for example, forpreserving human oocytes for in vitro fertilization.

Example 2 Cytotoxicity and Cryopreservation Screening

This example shows that compositions of the present invention havelittle to no cell toxicity and can achieve superior cryopreservationwhen compared to existing compounds, while reducing the necessary amountof CPAs and thus reducing CPA-associated toxicity.

Cytotoxicity Assays

In order to demonstrate the safety of cryoprotectant compositions of thepresent invention, a high-throughput cell-based cytotoxicity assay wasdeveloped utilizing the HEK 293 cell line, which is a sturdy and robuststem cell line grown from human embryonic kidney cells in tissueculture.

A Tecan Genesis Robotic Workstation was used to prepare solutions in 96-and 384-well plates. Solutions contained culture media, buffers, acryoprotectant composition of the present invention (Compound 12) orDMSO. Solutions were adjusted to the desired pH. Serial dilutions wereperformed to obtain solutions containing various concentrations ofCompound 12 and DMSO. Control experiments were performed using onlyculture media.

For these experiments, cells were seeded at low density (i.e., 10%confluence), exposed to solutions containing Compound 12 or DMSO, andplaced in a 37° C. incubator. The cells were allowed to grow untilcontrol cells that were treated only with empty vehicle approached 70%confluence (typically about 3 to 5 days). Assessment for compoundcytotoxicity was via MTT assay.

As can be seen in FIG. 7, the toxicity of Compound 12 did notsignificantly deviate from the that of culture media alone when analyzedby MTT assay. On the other hand, DMSO did not allow for warm survivalfor an extended period of time at any concentration above 0.5% (v/v).Notably, Compound 12 did not show toxicity at the concentrations inwhich it can prevent ice formation in a non-biological sample (0.5% w/v)and did not show significant toxicity at concentrations four timesgreater than this concentration, either.

These results show that compositions of the present invention wereeffective at ice-prevention even at concentrations where DMSO toxicitysignificantly reduced cell survival.

Cryopreservation Assays

Initial cryopreservation assays were performed using very simplesolutions, with and without the addition of Compound 12, in order tominimize confounding outside factors. For this first set of experiments,two sample solutions were prepared. The first sample solution containedsimple buffer and ethylene glycol (EG) at a concentration of 22.5%(v/v), and the second sample solution contained simple buffer, EG (22.5%(v/v)), and 5 mg/mL (0.5% (w/v)) of Compound 12.

HEK 293 cells were grown until 70% confluent, then treated with trypsinto remove adhesion proteins and yield free floating cells. Cells werecounted using a hemocytometer and sample cell concentrations wereadjusted to final concentrations of 10,000 cells per microliter. Cellswere then compressed into tight pellets by centrifugation, and eachsample was subsequently mixed with 20 μL of one of the sample solutions.Samples were then flash frozen by immersion in liquid nitrogen, followedby rewarming in a 37° C. water bath. After the freeze-thaw process,cells were suspended in a 400× volume of culture media for recovery. Thepositive control sample was treated with culture media at 37° C. and notsubjected to the freeze-thaw process. The negative control sample wastreated with culture media only during the freeze-thaw process. Afterrecovery, cells were stained with Calcein AM for 30 minutes and cellviability was measured using a fluorescence plate reader.

As shown in FIG. 8, the addition of Compound 12 greatly improved cellsurvival and demonstrated the ability of this compound to cryopreservecells. It was observed that the sample containing Compound 12 achievedcomplete vitrification without ice formation during the freezingprocess. In addition, the process of devitrification was bypassed muchmore rapidly compared to the sample lacking Compound 12.

A second set of experiments was performed to evaluate thecryopreservation potential of a formulation that contained 5 mg/mL ofCompound 12 plus a mixture of glycols, disaccharides, and a generalbuffer. Post-thaw survival following vitrification in liquid nitrogenwas evaluated as described above. As can be seen in FIG. 9, theformulation achieved near 100% (i.e., 98%) post-thaw survival of thecells, which was similar to the control group that was not exposed tofreezing treatment. The cell morphologies and florescence signals lookedidentical to the non-frozen controls, which indicated that little damageoccurred to the cells during the experiment.

As part of the second set of experiments, the cryopreservation potentialof the formulation was compared to two known cryopreservation reagents.VS2E is a DMSO-free and serum-free solution containing non-chemicallydefined polymers (see, e.g., Nishigaki et al. Int. J. Dev. Biol.55:3015-311 (2011)), and M22 is an organ vitrification solutionavailable from 21^(st) Century Medicine. FIG. 9 shows that theformulation containing Compound 12 achieved superior cryopreservation,as cell survival was 72% and 51% for VS2E and M22, respectively. Itshould be noted that for the M22 sample, background fluorescence mayhave skewed this result, as a count of live cells in the image suggestedthat far fewer than 51% of the cells had survived.

The compositions of the present invention were highly effective atpreventing ice formation in solutions containing significantly reducedethylene glycol. In particular, low concentrations of the compositions(e.g., 0.5% (w/v)) were sufficient to block ice growth duringvitrification and to keep solutions in a liquid, ice-free state on the20 uL scale, which is a scale that is useful for the preservation ofvarious types of cells.

In summary, these results show that compositions of the presentinvention can achieve superior cryopreservation and reduce the necessaryamount of CPAs, thus reducing cell toxicity that is associated withCPAs. The superior properties of the compositions of the presentinvention are especially useful for the treatment of particularlysensitive cell lines and/or when cells need to be cultured for longertime periods.

V. EXEMPLARY EMBODIMENTS

Exemplary embodiments provided in accordance with the presentlydisclosed subject matter include, but are not limited to, the claims andthe following embodiments:

1. A peptoid polymer according to formula (I):

a tautomer thereof or stereoisomer thereof,

wherein:

each R1 is independently selected from the group consisting of H,optionally substituted C1-18 alkyl, optionally substituted C2-18alkenyl, optionally substituted C2-18 alkynyl, optionally substitutedC1-18 hydroxyalkyl, optionally substituted alkoxy, optionallysubstituted C₁₋₁₈ alkylamino, optionally substituted C1-18 alkylthio,optionally substituted carboxyalkyl, C3-10 cycloalkyl, heterocycloalkyl,aryl, heteroaryl, (C3-10 cycloalkyl)alkyl, (heterocycloalkyl)alkyl,arylalkyl, and heteroarylalkyl,

wherein at least one instance of R1 is C1-18 hydroxyalkyl, and

wherein any of the cycloalkyl, heterocycloalkyl, aryl, and heteroarylgroups is optionally and independently substituted with one or more R3groups;

each R2 is independently selected from the group consisting of H,optionally substituted C1-18 alkyl, optionally substituted C2-18alkenyl, optionally substituted C2-18 alkynyl, optionally substitutedC1-18 hydroxyalkyl, optionally substituted C1-18 alkylamino, optionallysubstituted C1-18 alkylthio, and optionally substituted carboxyalkyl;

each R3 is independently selected from the group consisting of halogen,oxo, thioxo, OH, SH, amino, C1-8 alkyl, C1-8 hydroxyalkyl, C1-8alkylamino, and C1-8 alkylthio;

X and Y are independently selected from the group consisting of H,optionally substituted C1-8 alkyl, optionally substituted C1 8 acyl,optionally substituted C1-8 alkylamino, OH, SH, NH₂, carboxy, optionallysubstituted C1-8 hydroxyalkyl, optionally substituted C1-8 alkylamino,optionally substituted C2-8 alkylthio, optionally substituted C1-8carboxyalkyl, and halogen, or

alternatively X and Y are taken together to form a covalent bond; and

the subscript n, representing the number of monomers in the polymer, isbetween 2 and 50;

provided that all instances of R1 are not hydroxyethyl when n is between3 and 7.

2. The peptoid polymer of embodiment 1, wherein each R1 is independentlyselected from the group consisting of

wherein:

m is between 1 and 8; and

R3 is selected from the group consisting of H, C₁₋₈ alkyl, halogen,hydroxyl, thiol, nitro, amine, oxo, and thioxo.

3. The peptoid polymer of embodiment 2, wherein one or more R1 has astructure according to R1a:

4. The peptoid polymer of embodiment 3, wherein each R1a group isindependently selected from:

5. The peptoid polymer of embodiment 3, wherein each R1a group is:

6. The peptoid polymer of embodiment 3, wherein each R1a group is:

7. The peptoid polymer of embodiment 2, wherein one or more R1 has astructure according to R1b:

8. The peptoid polymer of embodiment 7, wherein each R1b group isindependently selected from:

9. The peptoid polymer of embodiment 7, wherein each R1b group is:

10. The peptoid polymer of embodiment 7, wherein each R1b group is:

11. The peptoid polymer of embodiment 1, wherein each R1 isindependently selected from the group consisting of

12. The peptoid polymer of embodiment 1, wherein each instance of R1 isan independently selected C1-18 hydroxyalkyl group.

13. The peptoid polymer of embodiment 12, wherein each instance of R1 isan independently selected C1-6 hydroxyalkyl group.

14. The peptoid polymer of embodiment 13, wherein each instance of R1 isthe same C1-6 hydroxyalkyl group.

15. The peptoid polymer of embodiment 14, wherein each instance of R1 is

16. The peptoid polymer of any one of embodiments 1 to 15, wherein eachinstance of R2 is H.

17. The peptoid polymer of any one of embodiments 1 to 16, wherein n isbetween 3 and 25.

18. The peptoid polymer of any one of embodiments 1 to 16, wherein n isbetween 8 and 50.

19. The peptoid polymer of any one of embodiments 1 to 16, wherein n isbetween 8 and 20.

20. The peptoid polymer of any one of embodiments 1 to 19, wherein X isselected from the group consisting of H, C₁₋₈ alkyl, and C₁₋₈ acyl; andY is selected from the group consisting of —OH and amino.

21. The peptoid polymer of any one of embodiments 1 to 19, wherein X andY are taken together to form a covalent bond.

22. The peptoid polymer of embodiment 1, wherein n is 10 and the peptoidpolymer comprises:

3 Nhp (2-((2-hydroxypropyl)amino)acetic acid) monomers and 7 Nsb(2-(sec-butylamino)acetic acid) monomers; or

4 Nhp monomers and 6 Nsb monomers; or

5 Nhp monomers and 5 Nsb monomers; or

6 Nhp monomers and 4 Nsb monomers; or

7 Nhp monomers and 3 Nsb monomers; or

8 Nhp monomers and 2 Nsb monomers; or

10 Nhp monomers.

23. The peptoid polymer of embodiment 22, wherein the peptoid polymerhas the sequence Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp; X is H or C1-8acyl; and Y is —OH or —NH₂ or C1-8alkyl.

24. The peptoid polymer of embodiment 22, wherein the peptoid polymerhas the sequence Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb; X is H or C1-8acyl; and Y is —OH or —NH₂ or C1-8alkyl.

25. The peptoid polymer of embodiment 22, wherein the peptoid polymerhas the sequence Nsb-Nhp-Nhp-Nhp-Nsb-Nhp-Nhp-Nhp-Nsb-Nhp; X is H or C1-8acyl; and Y is —OH or —NH₂ or C1-8alkyl.

26. The peptoid polymer of embodiment 22, wherein the peptoid polymerhas the sequence Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb; X is H or C1-8acyl; and Y is —OH or —NH₂ or C1-8alkyl.

27. The peptoid polymer of embodiment 22, wherein the peptoid polymerhas the sequence Nsb-Nhp-Nhp-Nhp-Nhp-Nhp-Nsb-Nhp-Nhp-Nhp; X is H or C1-8acyl; and Y is —OH or —NH₂ or C1-8alkyl.

28. The peptoid polymer of embodiment 22, wherein the peptoid polymerhas a sequence set forth in Table 2.

29. The peptoid polymer of embodiment 1, wherein n is 10 and the peptoidpolymer comprises:

3 Nhp (2-((2-hydroxypropyl)amino)acetic acid) monomers and 7 Nme(2-(methylamino)acetic acid) monomers; or

4 Nhp monomers and 6 Nme monomers; or

5 Nhp monomers and 5 Nme monomers; or

6 Nhp monomers and 4 Nme monomers; or

7 Nhp monomers and 3 Nme monomers; or

8 Nhp monomers and 2 Nme monomers.

30. The peptoid polymer of embodiment 29, wherein the peptoid polymerhas a sequence set forth in Table 3.

31. The peptoid polymer of embodiment 1, wherein n is 10 and the peptoidpolymer comprises:

5 Nhe (2-((2-hydroxyethyl)amino)acetic acid) monomers and 5 Nsb(2-(sec-butylamino)acetic acid) monomers; or

5 Nhp (2-((2-hydroxypropyl)amino)acetic acid) monomers and 5 Nbu(2-butylamino)acetic acid) monomers.

32. The peptoid polymer of embodiment 31, wherein the peptoid polymerhas a sequence set forth in Table 4.

33. The peptoid polymer of embodiment 1, wherein n is 10 and the peptoidpolymer comprises:

4 Nhp (2-((2-hydroxypropyl)amino)acetic acid) monomers and 6 Nib(2-(isobutylamino)acetic acid) monomers; or

4 Nhp monomers and 6 Nbu (2-butylamino)acetic acid) monomers; or

4 Nhp monomers and 6 Npr (2-propylamino)acetic acid) monomers; or

4 Nhp monomers and 6 Nip (2-(isopropylamino)acetic acid) monomers.

34. The peptoid polymer of embodiment 33, wherein the peptoid polymerhas a sequence set forth in Table 5.

35. The peptoid polymer of embodiment 1, wherein n is 14 and the peptoidpolymer comprises:

6 Nhp (2-((2-hydroxypropyl)amino)acetic acid) monomers and 8 Nsb(2-(sec-butylamino)acetic acid) monomers; or

7 Nhp monomers and 7 Nsb monomers; or

8 Nhp monomers and 6 Nsb monomers; or

10 Nhp monomers and 4 Nsb monomers; or

14 Nhp monomers.

36. The peptoid polymer of embodiment 35, wherein the peptoid polymerhas a sequence set forth in Table 6.

37. The peptoid polymer of embodiment 1, wherein n is 14 and the peptoidpolymer comprises:

6 Nhp (2-((2-hydroxypropyl)amino)acetic acid) monomers and 8 Nib(2-(isobutylamino)acetic acid) monomers; or

7 Nhp monomers and 7 Nib monomers; or

8 Nhp monomers and 6 Nib monomers; or

10 Nhp monomers and 4 Nib monomers; or

14 Nhp monomers.

38. The peptoid polymer of embodiment 37, wherein the peptoid polymerhas a sequence set forth in Table 7.

39. The peptoid polymer of embodiment 1, wherein n is 16 and the peptoidpolymer comprises:

5 Nhp (2-((2-hydroxypropyl)amino)acetic acid) monomers and 11 Nsb(2-(sec-butylamino)acetic acid) monomers; or

7 Nhp monomers and 9 Nsb monomers; or

8 Nhp monomers and 8 Nsb monomers; or

10 Nhp monomers and 6 Nsb monomers; or

12 Nhp monomers and 4 Nsb monomers; or

16 Nhp monomers.

40. The peptoid polymer of embodiment 39, wherein the peptoid polymerhas a sequence set forth in Table 8.

41. The peptoid polymer of embodiment 1, wherein n is 22 and the peptoidpolymer comprises:

7 Nhp (2-((2-hydroxypropyl)amino)acetic acid) monomers and 15 Nsb(2-(sec-butylamino)acetic acid) monomers; or

10 Nhp monomers and 12 Nsb monomers; or

11 Nhp monomers and 11 Nsb monomers; or

14 Nhp monomers and 8 Nsb monomers; or

17 Nhp monomers and 5 Nsb monomers; or

22 Nhp monomers.

42. The peptoid polymer of embodiment 41, wherein the peptoid polymerhas a sequence set forth in Table 9.

43. The peptoid polymer of any one of embodiments 1 to 42, wherein thepeptoid polymer forms a helical structure.

44. The peptoid polymer of any one of embodiments 1 to 43, wherein thepeptoid polymer reduces or inhibits ice crystal formation at atemperature within about 0° C. to about −20° C.

45. The peptoid polymer of any one of embodiments 1 to 43, wherein thepeptoid polymer reduces or inhibits ice crystal formation at atemperature within about −20° C. to about −40° C.

46. The peptoid polymer of any one of embodiments 1 to 43, wherein thepeptoid polymer reduces or inhibits ice crystal formation at about −20°C.

47. The peptoid polymer of any one of embodiments 1 to 43, wherein thepeptoid polymer reduces or inhibits ice crystal formation at atemperature within about −40° C. to about −200° C.

48. A peptoid-peptide hybrid comprising a peptoid polymer of any one ofembodiments 1 to 47 and one or more amino acids, wherein the one or moreamino acids are located at one or both ends of the peptoid polymerand/or between one or more peptoid monomers.

49. The peptoid-peptide hybrid of embodiment 48, wherein the one or moreamino acids are selected from the group consisting of alanine, cysteine,aspartic acid, glutamic acid, phenylalanine, glycine, histidine,isoleucine, arginine, lysine, leucine, methionine, asparagine, proline,glutamine, serine, threonine, valine, tryptophan, tyrosine, and acombination thereof.

50. The peptoid-peptide hybrid of embodiment 48, wherein the one or moreamino acids are selected from the group consisting of isoleucine,leucine, serine, threonine, alanine, valine, arginine, and a combinationthereof.

51. A cryoprotectant solution comprising a peptoid polymer of any one ofembodiments 1 to 47, a peptoid-peptide hybrid of any one of embodiments48 to 50, or a combination thereof.

52. The cryoprotectant solution of embodiment 51, further comprising acompound selected from the group consisting of an ionic species, apenetrating cryoprotectant, a non-penetrating cryoprotectant, anantioxidant, a cell membrane stabilizing compound, an aquaporin or otherchannel forming compound, an alcohol, a sugar, a sugar derivative, anonionic surfactant, a protein, dimethyl sulfoxide (DMSO), polyethyleneglycol (PEG), Ficoll®, polyvinylpyrrolidone, polyvinyl alcohol,hyaluronan, formamide, a natural or synthetic hydrogel, and acombination thereof.

53. The cryoprotectant solution of embodiment 52, wherein the alcohol isselected from the group consisting of propylene glycol, ethylene glycol,glycerol, methanol, butylene glycol, adonitol, ethanol, trimethyleneglycol, diethylene glycol, polyethylene oxide, erythritol, sorbitol,xythyritol, polypropylene glycol, 2-methyl-2,4-pentanediol (MPD),mannitol, inositol, dithioritol, 1,2-propanediol, and a combinationthereof.

54. The cryoprotectant solution of embodiment 52, wherein the sugar is amonosaccharide.

55. The cryoprotectant solution of embodiment 54, wherein themonosaccharide is selected from the group consisting of glucose,3-O-Methyl-D-glucopyranose, galactose, arabinose, fructose, xylose,mannose, and a combination thereof.

56. The cryoprotectant solution of embodiment 52, wherein the sugar is adisaccharide.

57. The cryoprotectant solution of embodiment 56, wherein thedisaccharide is selected from the group consisting of sucrose,trehalose, lactose, maltose, and a combination thereof.

58. The cryoprotectant solution of embodiment 52, wherein the sugar is apolysaccharide.

59. The cryoprotectant solution of embodiment 58, wherein thepolysaccharide is selected from the group consisting of raffinose,dextran, and a combination thereof.

60. The cryoprotectant solution of embodiment 52, wherein the PEG has anaverage molecular weight less than about 1,000 g/mol.

61. The cryoprotectant solution of embodiment 52, wherein the PEG has anaverage molecular weight between about 200-400 g/mol.

62. The cryoprotectant solution of embodiment 52, wherein the protein isselected from the group consisting of egg albumin, bovine serum albumin,human serum albumin, gelatin, and a combination thereof.

63. The cryoprotectant solution of embodiment 52, wherein the natural orsynthetic hydrogel comprises chitosan, hyaluronic acid, or a combinationthereof.

64. The cryoprotectant solution of embodiment 52, wherein the nonionicsurfactant is selected from the group consisting of polyoxyethylenelauryl ether, polysorbate 80, and a combination thereof.

65. A method for preserving a tissue, organ, or cell, the methodcomprising contacting the tissue, organ, or cell with a peptoid polymerof any one of embodiments 1 to 47, a peptoid-peptide hybrid of any oneof embodiments 48 to 50, a cryoprotectant solution of any one ofembodiments 51 to 64, or a combination thereof.

66. The method of embodiment 65, wherein the peptoid polymer,peptoid-peptide hybrid, cryoprotectant solution, or combination thereofis present in an amount sufficient to reduce or inhibit ice crystalformation at a temperature within about 0° C. to about −20° C.

67. The method of embodiment 65, wherein the peptoid polymer,peptoid-peptide hybrid, cryoprotectant solution, or combination thereofis present in an amount sufficient to reduce or inhibit ice crystalformation at a temperature within about −20° C. to about −40° C.

68. The method of embodiment 65, wherein the peptoid polymer,peptoid-peptide hybrid, cryoprotectant solution, or combination thereofis present in an amount sufficient to reduce or inhibit ice crystalformation at about −20° C.

69. The method of embodiment 65, wherein the peptoid polymer,peptoid-peptide hybrid, cryoprotectant solution, or combination thereofis present in an amount sufficient to reduce or inhibit ice crystalformation at a temperature within about −40° C. to about −200° C.

70. The method of any one of embodiments 65 to 69, wherein the tissue isa bioengineered tissue.

71. The method of any one of embodiments 65 to 70, wherein the peptoidpolymer, the peptoid-peptide hybrid, or a combination thereof is presentin amount between about 100 nM and about 100 mM.

72. The method of any one of embodiments 65 to 71, wherein the tissue,organ, or cell is selected from the group consisting of heart, liver,lung, kidney, pancreas, intestine, thymus, cornea, nerve cells, bloodplatelets, sperm cells, oocytes, embryonic cells, stem cells, humanpluripotent stem cells, hematopoietic stem cells, lymphocytes,granulocytes, immune system cells, bone cells, organoids, and acombination thereof.

73. A method for preserving a biological macromolecule, the methodcomprising contacting the biological macromolecule with a peptoidpolymer of any one of embodiments 1 to 47, a peptoid-peptide hybrid ofany one of embodiments 48 to 50, a cryoprotectant solution of any one ofembodiments 51 to 64, or a combination thereof.

74. The method of embodiment 73, wherein the biological macromolecule isselected from the group consisting of a nucleic acid, an amino acid, aprotein, an isolated protein, a peptide, a lipid, a composite structure,and a combination thereof.

75. A cosmetic care product comprising a peptoid polymer of any one ofembodiments 1 to 47, a peptoid-peptide hybrid of any one of embodiments48 to 50, a cryoprotectant solution of any one of embodiments 51 to 64,or a combination thereof.

76. An antifreeze product comprising a peptoid polymer of any one ofembodiments 1 to 47, a peptoid-peptide hybrid of any one of embodiments48 to 50, a cryoprotectant solution of any one of embodiments 51 to 64,or a combination thereof.

77. The antifreeze product of embodiment 76, wherein the antifreezeproduct is a deicing or ice-inhibiting product used to prevent, inhibit,or delay the formation of ice on an object.

78. The antifreeze product of embodiment 77, wherein the object isselected from the group consisting of an aircraft or a part thereof, agas pipeline, a window, electrical equipment, a drone, a cable, a powerline, mechanical equipment, a car engine, a gear system, and a brakesystem.

79. A frozen food product comprising a peptoid polymer of any one ofembodiments 1 to 47, a peptoid-peptide hybrid of any one of embodiments48 to 50, a cryoprotectant solution of any one of embodiments 51 to 64,or a combination thereof.

80. The frozen food product of embodiment 79, wherein the frozen foodproduct is selected from the group consisting of ice cream, yogurt,seafood, fruit, and meat products.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Nothing in thisspecification should be considered as limiting the scope of the presentinvention. All examples presented are representative and non-limiting.The above-described embodiments of the invention may be modified orvaried, without departing from the invention, as appreciated by thoseskilled in the art in light of the above teachings. It is therefore tobe understood that, within the scope of the claims and theirequivalents, the invention may be practiced otherwise than asspecifically described. All publications, patents, and patentapplications cited in this specification are herein incorporated byreference as if each individual publication, patent, or patentapplication were specifically and individually indicated to beincorporated by reference.

VI. INFORMAL SEQUENCE LISTING

SEQ ID NO: Sequence Notes  1 Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-NsbCompound 1  2 Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp Compound 10  3Nep-Nep-Xaa-Xaa-Xaa-Xaa-Nep-Nep-Nep-Nep-Nme-Nme Peptoid-Peptide Hybrid 4 Nme-Nme-Xaa-Nme-Nme-Nme-Nme-Nhp-Nhp-Nsb-Xaa- Peptoid-Peptide HybridNme-Nme-Xaa-Nme-Nme-Nme  5 Nme-Nme-Xaa-Nme-Nme-Nme-Nme-Nme-Nme-Nme-Peptoid-Peptide Hybrid Xaa-Xaa  6Arg-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb Peptoid-Peptide Hybrid(Compound 58)  7 Nsb-Nhp-Nhp-Nhp-Nsb-Nhp-Nhp-Nhp-Nsb-Nhp Compound 6  8Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb Compound 12  9Nsb-Nhp-Nhp-Nhp-Nhp-Nhp-Nsb-Nhp-Nhp-Nhp Compound 8 10Nsb-Nsb-Nsb-Nhp-Nhp-Nhp-Nsb-Nsb-Nsb-Nhp Compound 2

1. A peptoid polymer according to formula (I):

a tautomer thereof or stereoisomer thereof, wherein: each R¹ isindependently selected from the group consisting of optionallysubstituted C₁₋₁₈ alkyl, optionally substituted C₂₋₁₈ alkenyl,optionally substituted C₂₋₁₈ alkynyl, optionally substituted C₁₋₁₈hydroxyalkyl, optionally substituted alkoxy, optionally substitutedC₁₋₁₈ alkylamino, optionally substituted C₁₋₁₈ alkylthio, optionallysubstituted carboxyalkyl, C₃₋₁₀ cycloalkyl, heterocycloalkyl, aryl,heteroaryl, (C₃₋₁₀ cycloalkyl)alkyl, (heterocycloalkyl)alkyl, arylalkyl,and heteroarylalkyl, wherein at least one instance of R¹ is anoptionally substituted C₁₋₁₈ hydroxyalkyl group, and wherein any of thecycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups is optionallyand independently substituted with one or more R³ groups; each R² isindependently selected from the group consisting of H, optionallysubstituted C₁₋₁₈ alkyl, optionally substituted C₂₋₁₈ alkenyl,optionally substituted C₂₋₁₈ alkynyl, optionally substituted C₁₋₁₈hydroxyalkyl, optionally substituted C₁₋₁₈ alkylamino, optionallysubstituted C₁₋₁₈ alkylthio, and optionally substituted carboxyalkyl;each R³ is independently selected from the group consisting of halogen,oxo, thioxo, —OH, —SH, amino, C₁₋₈ alkyl, C₁₋₈ hydroxyalkyl, C₁₋₈alkylamino, and C₁₋₈ alkylthio; X and Y are independently selected fromthe group consisting of H, optionally substituted C₁₋₈ alkyl, optionallysubstituted C₁₋₈ acyl, optionally substituted C₁₋₈ alkylamino, —OH, —SH,—NH₂, carboxy, optionally substituted C₁₋₈ hydroxyalkyl, optionallysubstituted C₁₋₈ alkylamino, optionally substituted C₂₋₈ alkylthio,optionally substituted C₁₋₈ carboxyalkyl, and halogen, or alternativelyX and Y are taken together to form a covalent bond; and the subscript n,representing the number of monomers in the polymer, is between 6 and 50;provided that all instances of R¹ are not hydroxyethyl when n is between6 and
 7. 2. The peptoid polymer of claim 1, wherein each R¹ isindependently selected from the group consisting of

wherein: m is between 1 and 8; and R³ is selected from the groupconsisting of H, C₁₋₈ alkyl, halogen, hydroxyl, thiol, nitro, amine,oxo, and thioxo. 3-6. (canceled)
 7. The peptoid polymer of claim 2,wherein one or more R¹ has a structure according to R^(1b):

8-10. (canceled)
 11. The peptoid polymer of claim 1, wherein each R¹ isindependently selected from the group consisting of


12. The peptoid polymer of claim 1, wherein at least 2 instances of R¹are independently selected optionally substituted C₁₋₁₈ hydroxyalkylgroups.
 13. The peptoid polymer of claim 1, wherein at least 4 instancesof R¹ are independently selected optionally substituted C₁₋₁₈hydroxyalkyl groups.
 14. The peptoid polymer of claim 1, wherein theC₁₋₁₈ hydroxyalkyl group is an independently selected optionallysubstituted C₁₋₆ hydroxyalkyl group.
 15. (canceled)
 16. The peptoidpolymer of claim 1, wherein each instance of R² is H.
 17. The peptoidpolymer of claim 1, wherein n is between 6 and
 25. 18. (canceled) 19.The peptoid polymer of claim 1, wherein n is between 6 and
 22. 20. Thepeptoid polymer of claim 1, wherein X is selected from the groupconsisting of H, C₁₋₈ alkyl, and C₁₋₈ acyl; and Y is selected from thegroup consisting of —OH and amino.
 21. The peptoid polymer of claim 1,wherein X and Y are taken together to form a covalent bond. 22-43.(canceled)
 44. The peptoid polymer of claim 1, wherein the peptoidpolymer reduces or inhibits ice crystal formation at a temperaturewithin about 0° C. to about −20° C.
 45. (canceled)
 46. (canceled) 47.The peptoid polymer of claim 1, wherein the peptoid polymer reduces orinhibits ice crystal formation at a temperature within about −20° C. toabout −200° C. 48-50. (canceled)
 51. A cryoprotectant solutioncomprising a peptoid polymer of claim
 1. 52-64. (canceled)
 65. A methodfor preserving a biological sample, the method comprising contacting thebiological sample with a peptoid polymer of claim
 1. 66. The method ofclaim 65, wherein the biological sample comprises a tissue, organ, orcells. 67-69. (canceled)
 70. The method of claim 66, wherein the tissueis a bioengineered tissue.
 71. (canceled)
 72. The method of claim 66,wherein the tissue or organ is selected from the group consisting ofheart, liver, lung, kidney, pancreas, intestine, thymus, cornea, bonemarrow, organoids, and a combination thereof.
 73. A method forpreserving a biological macromolecule, the method comprising contactingthe biological macromolecule with a peptoid polymer of claim
 1. 74. Themethod of claim 73, wherein the biological macromolecule is selectedfrom the group consisting of a nucleic acid, an amino acid, a protein,an isolated protein, a peptide, a lipid, a composite structure, and acombination thereof. 75-80. (canceled)
 81. The method of claim 66,wherein the cells are selected from the group consisting of heart cells,liver cells, lung cells, kidney cells, pancreatic cells, intestinalcells, induced pluripotent stem cells, neural cells, blood cells,hematopoietic stem cells, lymphocytes, granulocytes, immune systemcells, bone cells, stem cells, sperm cells, oocytes, embryonic cells,and a combination thereof.
 82. The method of claim 74, wherein thebiological macromolecule is a nucleic acid.
 83. The method of claim 82,wherein the nucleic acid is DNA or RNA.